Methods of treating metabolic syndrome related conditions using retinoic acid receptor agonists

ABSTRACT

This invention relates to pharmaceutical compositions and methods for treating (including managing) or preventing metabolic syndrome related conditions using one or more RARβ (e.g., RARβ2) agonists. Such conditions include, but are not limited to, diseases in pancreas, liver, kidney, testes, muscle, or adipose tissue, as well as other organs that are associated with high fat diet and/or vitamin A deficiency, as well as other conditions associated with abnormal level of triglyceride, cholesterol and/or glucose.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/112,159. Filed Jul. 15, 2016 which claims priority to National PhaseApplication of PCT International Application PCT/US15/11820, filed Jan.17, 2014 which is a Continuation in Part to PCT InternationalApplication PCT/US2014/012083, filed Jan. 17, 2014, which claimspriority to and the benefit of U.S. Provisional Application Ser. No.61/990,808, filed May 9, 2014. All applications above are incorporatedherein in their entirety.

GOVERNMENT FUNDING

This invention was made with Government support under Grant NumberDE010389 and CA043796 awarded by the National Institutes of Health. TheUnited States Government has certain rights in the invention.

FIELD

The invention relates to the treatment or prevention of certainmetabolic syndrome related conditions. For example, the inventionrelates to controlling the level cholesterol, triglyceride, and/orglucose in a subject in need thereof, as well as treating or preventingdiseases or conditions caused by fat accumulation or vitamin Adeficiency in a subject in need thereof.

BACKGROUND

Metabolic syndrome is caused by a cluster of metabolic risk factorswhich include, but are not limited to, insulin resistance, hypertension(high blood pressure), cholesterol abnormalities, and an increased riskfor blood clotting. Examples of metabolic syndrome related conditionsinclude vitamin deficiencies, diabetes, fatty liver, high bloodpressure, insulin resistance, obesity, abnormal cholesterol and/ortriglyceride levels, artery and heart diseases.

After smoking, high fat diet is said to be the second most lethal habit,causing 300,000 deaths each year in the U.S. alone. High fat diet leadsto many health problems, including obesity, stroke, cancer, high bloodpressure, diabetes, osteoarthritis, rheumatoid arthritis, multiplesclerosis, heart disease, and diseases in other organs such as liver andkidney.

Diabetes is a group of diseases characterized by high blood glucoselevels that result from defects in the body's ability to produce and/oruse insulin. In 2011 there were an estimated 366 million cases ofdiabetes worldwide, according to the International Diabetes Federation,and these cases are estimated to increase to 522 million by 2030 (1, 2).In the U.S. there were 23.7 million diagnosed cases, with an estimatedhealthcare cost of $113 billion (2, 3). Type II diabetes results wheninsulin-directed metabolism of glucose is impaired in peripheral tissuessuch as fat and muscle, and production of insulin by pancreatic β-cellscannot meet metabolic demands due to loss of β-cell number and function(4). In type I diabetes, auto-immune destruction of insulin-producingpancreatic β-cells gives rise to hyperglycemia (5). Each year in theUnited States there are over 30,000 new cases of type I diabetesdiagnosed (6). Patients with type I diabetes can control their bloodglucose level with insulin supplements (7). However, the differentiationof stem cells into pancreatic β-cells could be a long term, bettersolution (8-10).

Type II diabetes is more common. In early stages of type II diabetes thebody does not use insulin properly, a phenomenon known as insulinresistance. In response to insulin resistance the pancreas will makeextra insulin to make up for it. But over time there won't be enoughinsulin to keep blood glucose at normal levels because insulin-producingpancreatic β-cells will fail to cope with increasing demand leading totheir destruction and decreased function. Type II diabetes is anincreasingly prevalent disease that due to a high frequency ofcomplications leads to a significant reduction of life expectancy.Because of diabetes associated microvascular complications, type IIdiabetes is currently the most frequent cause of adult-onset loss ofvision, renal failure, and amputations in the industrialized world. Inaddition, the presence of type II diabetes is associated with a two tofive fold increase in cardiovascular disease risk. After long durationof disease, most patients with type II diabetes will eventually fail onoral therapy and become insulin dependent with the necessity for dailyinjections and multiple daily glucose measurements.

A third type of diabetes, gestational diabetes, is developed by manywomen usually around the 24th week of pregnancy. Treatment forgestational diabetes aims to keep blood glucose levels equal to those ofpregnant women who don't have gestational diabetes.

Some patients with diabetes can manage their conditions with healthyeating and exercise. Some will need to have prescribed medicationsand/or insulin to keep blood glucose levels. In addition, diabetes is aprogressive disease. Even if medication is not required at first, it maybe needed overtime.

Non-alcoholic fatty liver disease (NAFLD) is marked by lipidaccumulation in hepatocytes (steatosis) without evidence of hepatitis orliver fibrosis (69, 70). NAFLD is a major risk factor for development ofnon-alcohol steatohepatitis (NASH) and hepatocellular carcinoma (71).Driven by rising rates of obesity, diabetes and insulin resistance,NAFLD is currently the most common form of liver disease in the UnitedStates with an estimated 55 million cases (69). At the current rate,NAFLD will reach epidemic proportions in the United States by 2030; yetno FDA approved pharmacological therapy exist for prevention ortreatment of NAFLD (69).

Over the last decade, experimental animal and human data suggests thathepatic stellate cells (HSCs) are an important cellular target fordevelopment of pharmacological therapies for prevention or treatment ofNAFLD spectrum liver diseases (73). HSCs are star-like cells that residein the liver sinusoids whose main function are to store 80-90% of thetotal body vitamin A (VA) pool (74). During hepatic injury HSCs losingtheir VA storage capacity, trans-differentiate into myofibroblasts andorchestrate wound healing by secreting components of extra-cellularmatrix including type 1 collagen (col1a1) and alpha-smooth muscle actin(α-SMA) (72, 73). During pathogenesis of unchecked NAFLD, HSCsproliferate and become highly fibrotic through hyper-secretion col1a1and α-SMA leading to liver scarring and an inflammation cascade thatdrives further hepatic fibrosis and liver damage (72,73).

Diabetes is the most common cause of kidney failure, accounting fornearly 44 percent of new cases. Even when diabetes is controlled, thedisease can lead to Chronic Kidney Disease (CKD) and kidney failure.Nearly 24 million people in the United States have diabetes, and nearly180,000 people are living with kidney failure as a result of diabetes.People with kidney failure undergo either dialysis, an artificialblood-cleaning process, or transplantation to receive a healthy kidneyfrom a donor. In 2005, care for patients with kidney failure cost theUnited States nearly $32 billion.

Triglycerides are composed of glycerol and various fatty acids, whichare used to store energy and provide energy to muscles. Triglyceridesare the end product of digesting and breaking down fats in meals, butsome triglycerides are made in the body from other energy sources suchas carbohydrates. Normally only small amounts are found in the blood.Extra triglycerides are stored in different places of the body in casethey are needed later. High blood triglyceride levels (e.g., as inhypertriglyceridemia) have been linked to obesity, diabetes, and agreater chance for heart disease.

Cholesterol is a sterol, one of three major classes of lipids which allanimal cells make and utilize to construct their membranes. It is alsothe precursor of the steroid hormones, bile acids and vitamin D. Sincecholesterol is insoluble in water, it is transported in the blood plasmawithin protein particles (lipoproteins). Elevated serum cholesterollevels are a major risk factor for development of atherosclerosis,myocardial infarction, and ischemic stroke. Approximately 71 millionAmerican adults have significantly elevated cholesterol levels, andamong these adults only 1 out of 3 have this condition under control((CDC) CfDCaP., 2011, MMWR Morb Mortal Wkly Rep. 60(4):109-14).

In view of the health risks, the American Heart Association recommendsthat everyone over the age of 20 should get a lipid panel test tomeasure cholesterol and triglycerides at least every five years. Ahealthy diet and exercise plan can lower triglyceride levels, improvecholesterol, and lower the risk of heart disease and otherhypertriglyceridemia or hypercholesterolemia-associated diseases. Ittakes time for participants to lose body weight and improve their plasmatriglyceride and cholesterol levels. Often even after these levels arenormalized, the great majority of participants regain body weight andfall back to the previous hyperlipidemia and hypercholesterolemiaconditions when followed for 3-5 years. In certain conditions, such asfamilial hypercholesterolemia, medication or even surgery is required.

Drugs useful for the treatment of hypertriglyceridemia include fatabsorption inhibitors that block pancreatic triglyceride lipase in theintestine, thermogenic agents that increase basal metabolism rate, aswell as anorectics that suppress appetite. Drugs useful for thetreatment of hypercholesterolemia include inhibitors for cholesterolbiosynthesis (statins), cholesterol absorption inhibitors, bile acidsequestrants, fibric acid derivatives and high doses (3-6 g/day) ofniacin. Each of these drugs has its therapeutic limitations, and severeside-effects have been reported for some drugs. Current lipid loweringtherapies do not sufficiently address the high triglyceride andcholesterol levels that are now known to be an important risk factor forcardiovascular disease without unwanted side effects.

Despite of multiple drugs under investigation, the health risksassociated with high triglyceride and cholesterol levels are actuallyincreasing. A new guideline was recently issued by the American Collegeof Cardiology and the American Heart Association (ACC-AHA). This newguideline would increase the number of U.S. adults receiving or eligiblefor cholesterol control therapy from 43.2 million (37.5%) to 56.0million (48.6%), with most of this increase in numbers (10.4 million of12 8 million) would occur among adults who are without cardiovasculardisease but would be classified solely on the basis of their 10-yearrisk of a cardiovascular event (Pencina et al., 2014, N. Engl. J. Med.370 (15): 1422-1431). In addition, more than 50 million Americans arecurrently prescribed statins, but up to 20% of adults with elevatedcholesterol are unable to use statins due to side effects such as muscleachiness and weakness (Mampuya et al., 2013, Am Heart J.,166(3):597-603. doi: 10.1016/j.ahj.2013.06.004.).

Retinoids are structurally related to vitamin A (VA) and are used totreat dermatological disorders and some cancers (Tang et al., 2011, AnnuRev Pathol., 6:345-64. doi: 10.1146/annurev-pathol-011110-130303;Baldwin et al., 2013, J Drugs Dermatol., 12(6):638-42. PubMed PMID:23839179). Previously, numerous studies have demonstrated that a commonside effect of retinoid administration to humans and rodents is bothhypertriglyceridemia and hypercholesterolemia (Ellis et al., 1982, ArchDermatol., 118(8):559-62; Lyons et al., 1982, Br JDermatol.,107(5):591-5; Marsden J., 1986, Br J Dermatol., 114(4):401-7;Barth et al., 1993, Br J Dermatol., 129(6):704-7). Although elevatedserum lipid profiles of subjects on retinoid therapy revert back tobaseline levels upon cessation of treatment, these observations haveraised concern that retinoid (RA) therapy could increase risk forcardiovascular disease (Marsden J., 1986, Br J Dermatol., 114(4):401-7).Standeven et al. (1996, Fundamental and Applied Toxicology 33, 264-271)reported that retinoic acid receptors mediate retinoid-inducedhypertriglyceridemia in rats.

There is an unmet medical need for methods, medicaments andpharmaceutical compositions to treat (including managing) the abovemetabolic syndrome related conditions, particularly with regard totreatments having disease-modifying properties, rapid impact, and at thesame time showing a good safety profile. The present invention providescompositions and methods to meet the unmet medical needs.

SUMMARY

This invention discloses pharmaceutical compositions and methods fortreating (including managing) or preventing metabolic syndrome relatedconditions. Such conditions include, but are not limited to, diseases inpancreas, liver, kidney, testes, muscle, or adipose tissue, as well asother organs that are associated with high fat diet and/or vitamin Adeficiency, as well as other conditions associated with abnormal levelof triglyceride, cholesterol and/or glucose.

According to certain embodiments, the invention provides a method oftreating or preventing a disease in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist, where the disease is selected from the group consistingof diabetes, a cardiovascular disease, a liver disease, a kidneydisease, obesity, hyperlipidemia, hypertriglyceridemia, orhyperglycemia.

According to certain embodiments, the invention provides a method oftreating or preventing a pancreatic disease in a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

In certain embodiments, the pancreatic disease is associated withobesity.

In certain embodiments, the pancreatic disease is associated with a highfat diet.

In certain embodiments, the pancreatic disease is associated withvitamin A deficiency in the pancreas.

The pancreatic disease may be diabetes, which may be type I or type IIdiabetes, or gestational diabetes.

According to certain embodiments, the invention provides a method ofincreasing RARβ level in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

In certain embodiments, RARβ level is increased in an organ.

The organ may be pancreas, liver, kidney, testes, muscle, or adiposetissue.

According to certain embodiments, the invention provides a method oftreating or preventing the degeneration of pancreatic beta cells in asubject in need thereof comprising administering to the subject vitaminA or a retinoic acid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofmaintaining or improving the function of pancreatic beta cells in asubject in need thereof comprising administering to the subject vitaminA or a retinoic acid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling insulin secretion in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofmaintaining or improving pancreatic insulin secretion in a subject inneed thereof comprising administering to the subject vitamin A or aretinoic acid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling insulin sensitivity in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofmaintaining or improving insulin sensitivity in a subject in needthereof comprising administering to the subject vitamin A or a retinoicacid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling insulin metabolism in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofmaintaining or improving insulin metabolism in a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling insulin resistance in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

Vitamin A or a retinoic acid receptor-beta (RARβ) agonist maysimultaneously control insulin resistance and insulin secretionaccording to one embodiment of the invention. As such, the number oflarge pancreatic islets and/or pancreatic insulin content may be reducedin the subject in need thereof.

According to certain embodiments, the invention provides a method ofcontrolling the level of glucagon in a subject in need thereofcomprising administering to said subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofmaintaining or improving the level of glucagon in a subject in needthereof comprising administering to said subject vitamin A or a retinoicacid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method oftreating or preventing fat deposit of a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling body weight in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling inflammation of a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method oftreating or preventing inflammation of a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofdecreasing the level of an inflammatory mediator in a subject in needthereof comprising administering to the subject vitamin A or a retinoicacid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling oxidative stress in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofdecreasing oxidative stress in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

In certain embodiments, the production of the inflammatory mediator isdecreased.

In certain embodiments, the secretion of the inflammatory mediator isdecreased.

The inflammatory mediator may be monocyte chemotactic protein (mcp-1) ortumor necrosis factor alpha (tnf-α) according to certain embodiments.

In certain embodiments, the fat deposit, inflammation or oxidativestress is in an organ.

The organ may be pancreas, liver, kidney, testes, muscle, or adiposetissue.

According to certain embodiments, the invention provides a method oftreating or preventing a liver disease in a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

In certain embodiments, the liver disease is associated with obesity.

In certain embodiments, the liver disease is associated with a high fatdiet.

In certain embodiments, the liver disease is associated with vitamin Adeficiency.

In certain embodiments, the liver disease is fatty liver disease (FLD),liver fibrosis, or hepatic steatosis.

In certain embodiments, the liver disease is non-alcoholic FLD (NAFLD),alcohol associated FLD, or non-alcoholic steatohepatitis (NASH).

In certain embodiments, the liver disease is associated with reducedvitamin A level in the liver.

According to certain embodiments, the invention provides a method ofdecreasing the activation of hepatic stellate cells (HSCs) in a subjectin need thereof comprising administering to the subject vitamin A or aretinoic acid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofdecreasing the level of hepatic reactive oxygen species (ROS) in asubject in need thereof comprising administering to the subject vitaminA or a retinoic acid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofdecreasing the level of alpha smooth muscle actin (α-SMA) in a subjectin need thereof comprising administering to the subject vitamin A or aretinoic acid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofincreasing the level of lethicin:retinol acyltransferase (LRAT) in theliver of a subject in need thereof comprising administering to thesubject vitamin A or a retinoic acid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofincreasing the level of RARβ in the liver of a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofdecreasing the level of SRBP1c in the liver of a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

In certain embodiments, the subject has a liver disease.

In certain embodiments, the liver disease is fatty liver disease (FLD),liver fibrosis, or hepatic steatosis.

In certain embodiments, the liver disease is non-alcoholic FLD (NAFLD),alcohol associated FLD, or non-alcoholic steatohepatitis (NASH).

In certain embodiments, the liver disease is associated with reducedvitamin A level in the liver.

In certain embodiments, the liver disease is associated with a pancreasdisease.

According to certain embodiments, the invention provides a method oftreating or preventing a kidney disease in a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

In certain embodiments, the kidney disease is associated with obesity.

In certain embodiments, the kidney disease is associated with a high fatdiet.

In certain embodiments, the kidney disease is kidney fibrosis.

In certain embodiments, the kidney disease is a chronic kidney disease.

In certain embodiments, the kidney disease is diabetic nephropathy.

In certain embodiments, the kidney disease is associated with apancreatic disease.

In certain embodiments, the kidney disease is associated with a liverdisease.

In certain embodiments, the kidney disease is associated with reducedvitamin A level in the kidney.

According to certain embodiments, the invention provides a method ofincreasing the level of lethicin:retinol acyltransferase (LRAT) in thekidney of a subject in need thereof comprising administering to thesubject vitamin A or a retinoic acid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method oftreating or preventing a disease associated with an organ-specificvitamin A deficiency in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

In certain embodiments, the organ-specific vitamin A deficiency isassociated with obesity.

In certain embodiments, the organ-specific vitamin A deficiency isassociated with a high fat diet.

In certain embodiments, the subject has a normal serum level of vitaminA or retinyl esters.

In certain embodiments, the subject has an abnormal level of vitamin Aor retinyl esters in a non-serum sample.

The organ may be pancreas, liver, kidney, testes, muscle, or adiposetissue.

According to certain embodiments, the invention provides a method oftreating or preventing fibrosis in a subject in need thereof comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofdecreasing the accumulation of fat in a subject in need thereofcomprising administering to the subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist.

In certain embodiments, the fibrosis or accumulation of fat is in anorgan.

The organ may pancreas, liver, kidney, testes, muscle, or adiposetissue.

According to certain embodiments, the vitamin A or agonist of retinoicacid receptor-beta (RARβ) is administered three times daily.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) is administered at an amount from 30-200 mg perday.

In certain embodiments, the vitamin A or agonist is administered at anamount from 50-150 mg per day.

In certain embodiments, the vitamin A or agonist is administered at anamount from 50-100 mg per day/

In certain embodiments, the vitamin A or agonist is administered at anamount from 100-150 mg per day.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) is administered orally.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) is administered intravenously or subcutaneously.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) does not elevate serum triglyceride in the subject.

In certain embodiments, the vitamin A or agonist of retinoic acidreceptor-beta (RARβ) does not increase cardiovascular risk in thesubject.

In certain embodiments, a therapeutic effective amount of the vitamin Aor agonist of RARβ is administered.

In certain embodiments, both vitamin A and an agonist of RARβ are bothadministered to the subject.

In certain embodiments, vitamin A and an agonist of RARβ areadministered concomitantly.

In certain embodiments, vitamin A and an agonist of RARβ areadministered sequentially.

According to certain embodiments, the invention provides apharmaceutical composition comprising vitamin A or a retinoic acidreceptor-beta (RARβ) agonist or a pharmaceutically acceptable saltthereof at an amount from about 10 mg to about 60 mg.

In certain embodiments, the amount of the vitamin A or agonist is from15 mg to about 50 mg.

In certain embodiments, the amount of the vitamin A or agonist is from15 mg to about 35 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 35 mg to about 50 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 30 mg to about 200 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 50 mg to about 150 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 50 mg to about 100 mg.

In certain embodiments, the amount of the vitamin A or agonist is fromabout 100 mg to about 150 mg.

According to certain embodiments, the invention provides apharmaceutical composition comprising vitamin A or a retinoic acidreceptor-beta (RARβ) agonist or a pharmaceutically acceptable saltthereof at a concentration from about 0.1 mg to about 10 mg per 100 ml.

In certain embodiments, the concentration is from about 0.5 mg to about5 mg per 100 ml.

In certain embodiments, the concentration is from about 1 mg to about 3mg per 100 ml.

In certain embodiments, the concentration is from about 1.5 mg to about2.5 mg per 100 ml.

In certain embodiments, the agonist is a highly specific RARβ agonist.

In certain embodiments, the agonist is AC261066.

In certain embodiments, the agonist is AC55649.

In certain embodiments, the pharmaceutical composition comprises bothvitamin A and an agonist of RARβ.

According to certain embodiments, the invention provides a method ofcontrolling triglyceride level in a subject in need thereof, comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling cholesterol level in a subject in need thereof, comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method oftreating or preventing hypertriglyceridemia or a condition associatedwith hypertriglyceridemia in a subject in need thereof, comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method oftreating or preventing hypercholesterolemia or a condition associatedwith hypercholesterolemia in a subject in need thereof, comprisingadministering to the subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofreducing the production of HMG-CoA reductase in a subject in needthereof, comprising administering to the subject vitamin A or a retinoicacid receptor-beta (RARβ) agonist.

According to certain embodiments, the invention provides a method ofreducing a clinically significant side effect of elevated bloodtriglyceride and/or cholesterol level caused by a drug in a subject inneed thereof, comprising administering to the subject treated by saiddrug vitamin A or a retinoic acid receptor-beta (RARβ) agonist.

In certain embodiments, the triglyceride and/or cholesterol level in anorgan (e.g., pancreas, liver, kidney, testes, muscle, or adipose tissue)is controlled.

According to certain embodiments, the invention provides a method ofregulating the expression of genes involved in lipogenesis and lipidcatabolism.

In certain embodiments, the expression of such genes is regulated in anorgan (e.g., pancreas, liver, kidney, testes, muscle, or adiposetissue). The regulation may result in an increase of oxidation of lipidsor a decrease in lipogenesis.

According to certain embodiments, the invention provides a method ofcontrolling glucose level in a subject in need thereof, comprisingadministering to said subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

According to certain embodiments, the invention provides a method ofcontrolling glucose intolerance in a subject in need thereof, comprisingadministering to said subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

In certain embodiments, the glucose level in an organ (e.g., pancreas,liver, kidney, testes, muscle, or adipose tissue) is controlled.

The subject in need may have a metabolic syndrome related conditionaccording to one embodiment of the invention.

The subject in need may have a condition selected from the groupconsisting of diabetes, cardiovascular disease, hyperglycemia, andhyperlipidemia.

According to certain embodiments, the invention provides a method ofcontrolling insulin resistance in a subject in need thereof, comprisingadministering to said subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist.

The retinoic acid receptor-beta (RARβ) agonist of the present inventionmay reduce triglyceride or cholesterol synthesis in the subjectaccording to certain embodiments.

The retinoic acid receptor-beta (RARβ) agonist may reduce triglycerideor cholesterol transport in the subject according to certainembodiments.

The retinoic acid receptor-beta (RARβ) agonist is a highly specific RARβagonist according to certain embodiments.

The retinoic acid receptor-beta (RARβ) agonist is a highly specificRARβ2 agonist according to certain embodiments.

The retinoic acid receptor-beta (RARβ) agonist is AC201066 according tocertain embodiments.

The retinoic acid receptor-beta (RARβ) agonist is AC55649 according tocertain embodiments.

The condition associated with hypertriglyceridemia/hypercholesterolemiais diabetes according to certain embodiments.

The condition associated with hypertriglyceridemia/hypercholesterolemiais a cardiovascular disease according to certain embodiments.

The condition associated with hypertriglyceridemia/hypercholesterolemiais hyperlipidemia according to certain embodiments.

The agonist of retinoic acid receptor-beta (RARβ) is administered threetimes daily according to certain embodiments.

The retinoic acid receptor-beta (RARβ) agonist is administered at anamount from about 30 mg to about 200 mg per day according to certainembodiments.

The retinoic acid receptor-beta (RARβ) agonist is administered at anamount from about 50 mg to about 150 mg per day according to certainembodiments.

The retinoic acid receptor-beta (RARβ) agonist is administered at anamount from about 50 mg to about 100 mg per day according to certainembodiments.

The retinoic acid receptor-beta (RARβ) agonist is administered at anamount from about 100 mg to about 150 mg per day according to certainembodiments.

The retinoic acid receptor-beta (RARβ) agonist is administered orallyaccording to certain embodiments.

The retinoic acid receptor-beta (RARβ) agonist is administeredintravenously or subcutaneously according to certain embodiments.

The method may further comprise administering a second drug according tocertain embodiments.

The second drug is a drug for treating hypertriglyceridemia or acondition associated with hypertriglyceridemia according to certainembodiments.

The second drug is a drug for treating hypercholesterolemia or acondition associated with hypercholesterolemia according to certainembodiments.

The second drug is another retinoic acid receptor-beta (RARβ) agonistaccording to certain embodiments.

According to certain embodiments, the triglyceride level in the blood ofthe subject is reduced to be less than 150 mg/dL.

According to certain embodiments, the triglyceride level in the blood ofthe subject is reduced to be 150 to 199 mg/dL.

According to certain embodiments, the cholesterol level in the blood ofthe subject is reduced to be 200 mg/dL or less.

According to certain embodiments, the cholesterol level in the blood ofthe subject is reduced to be 201 to 240 mg/dL.

According to certain embodiments, the production of HMG-CoA reductase inthe subject is reduced at the mRNA level.

According to certain embodiments, the therapeutic effect of vitamin A ora retinoic acid receptor-beta (RARβ) agonist may be achieved from about1 day to about 8 days after the agonist is administered to the subjectin need.

According to certain embodiments, the invention provides apharmaceutical composition comprising vitamin A or a retinoic acidreceptor-beta (RARβ) agonist having at least 70% RARβ2 binding affinityof AC261066, or a pharmaceutically acceptable salt thereof at an amountfrom about 10 mg to about 60 mg.

The pharmaceutical composition may contain the agonist at an amount from15 mg to about 50 mg according to certain embodiments.

The pharmaceutical composition may contain the agonist at an amount from15 mg to about 35 mg according to certain embodiments.

The pharmaceutical composition may contain the agonist at an amount fromabout 35 mg to about 50 mg according to certain embodiments.

The pharmaceutical composition may contain the agonist at an amount fromabout 30 mg to about 200 mg according to certain embodiments.

The pharmaceutical composition may contain the agonist at an amount fromabout 50 mg to about 150 mg according to certain embodiments.

The pharmaceutical composition may contain the agonist at an amount fromabout 50 mg to about 100 mg according to certain embodiments.

The pharmaceutical composition may contain the agonist at an amount fromabout 100 mg to about 150 mg according to certain embodiments.

According to certain embodiments, the invention provides apharmaceutical composition comprising vitamin A or a retinoic acidreceptor-beta (RARβ) agonist or a pharmaceutically acceptable saltthereof at a concentration from about 0.1 mg to about 10 mg per 100 ml.

The pharmaceutical composition may contain the agonist at aconcentration from about 0.5 mg to about 5 mg per 100 ml according tocertain embodiments.

The pharmaceutical composition may contain the agonist at aconcentration from about 1 mg to about 3 mg per 100 ml according tocertain embodiments.

The pharmaceutical composition may contain the agonist at aconcentration from about 1.5 mg to about 2.5 mg per 100 ml according tocertain embodiments.

The pharmaceutical composition may further comprise another agonist ofRARβ according to certain embodiments.

The retinoic acid receptor-beta (RARβ) agonist of the pharmaceuticalcomposition is a highly specific RARβ2 agonist according to certainembodiments.

The retinoic acid receptor-beta (RARβ) agonist of the pharmaceuticalcomposition is AC261066 according to certain embodiments.

The retinoic acid receptor-beta (RARβ) agonist of the pharmaceuticalcomposition is AC55649 according to certain embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Pancreatic endocrine differentiation protocol and its impact onthe molecular level. (A) Schematic representation of the endocrinedifferentiation protocol adapted from D'Amour et al. (2006) used onmouse ES cells. Briefly, embryonic stem (ES) cells are treated withdifferent growth factors to successively differentiate into definitiveendoderm (DE), pancreatic progenitor (PP), endocrine progenitor (EP),and endocrine cells (EC). (B) WT mouse ES cells were subjected to the17-day differentiation protocol. Each lane represents a differentcondition at specific time points. RT-PCR analyses were performed tomonitor the expression of pancreatic differentiation markers such asinsulin-1 (Ins1), glucagon (Gcg), somatostatin (Sst), neurogenin-3(Ngn3), Pdx1 and Sox17, as well as the stem cell markers Nanog and Rex1.HPRT1 amplification was used as a control housekeeping gene. Pancreasextracts from C57BL/6 WT mice were used as a positive control.

FIG. 2: Impact of RARβ deletion on Pdx1 expression through pancreaticendocrine differentiation process. (A) RT-PCR analysis confirming thesuppression of RARβ in KO ES cells. Analysis of Cyp26a1, a RA-responsivegene, demonstrates the presence of RA signaling activity via otherreceptors in RARβ KO cells. HPRT1 was used as a control housekeepinggene. (B) Indirect immunofluorescence staining for Pdx1 (green) in WTand RARβ KO, at 5, 11, 14, and 17 days in the absence (untreated) or inthe presence (treated) of growth factors used in the differentiationprotocol. Cells were counterstained using rhodamine-conjugatedphalloidin, which binds to F-actin (red) and nuclei were stained withDAPI (blue) (Bars=50 μm).

FIG. 3: Expression of pancreatic differentiation markers in WT and RARβknockout (KO) ES cells. Transcript expression analyses of (A) early, (B)mid, and (C) late stage endocrine pancreatic differentiation markers inWT and RARβ KO ES cells. RT-PCR amplification of (A) Nanog, Ngn3, (B)Pax6, Isl-1, and (C) Ins1, Gcg, and Iapp mRNA was performed in both celllines at 5, 11, 14, and 17 days of the differentiation protocol. In eachcase, RARβ expression was monitored in both cell lines and HPRT1 wasused as a control housekeeping gene. Relative amounts, normalized toHPRT1 levels for each marker tested, are shown in histograms (n=3; *:p≤0.05; **: p≤0.0079; ***: p≤0.0003).

FIG. 4: In vivo characterization of RARβ deletion on islets ofLangerhans functionality and glucose metabolism. (A) Indirectimmunofluorescence staining of C-peptide (green) and Glucagon (red) onC57BL/6 WT and RARβ KO mouse pancreas tissue sections. Pancreaticislet-corresponding regions were circled by dashed lines and nuclei weremarked with DAPI (blue) (bars=50 μm). Islet size were quantified persurface area units (cm²), with respect to high resolution micrographs,for each group and presented as histogram (n=6; **: p=0.031). Westernblot analysis of C-peptide and Glucagon expression was performed on WTand RARβ KO mouse pancreas protein extracts. Ins-1 cells were used aspositive control for C-peptide expression, while immunodetection ofactin was used as a loading control. (B) Blood glucose concentration(mg/dL) in WT and RARβ null, knockout mice after 15 h fasting (left)(n≥5; p=0.0011). Blood glucose clearance (right) for WT (♦) and RARβ KO(▪) mice was measured following a 2 mg/kg dextrose i.p. injection.Relative blood glucose levels were assessed at 0, 15, 30, 45, 60, and120 minutes post-injection (n≥6; *: p=0.0137; **: p≤0.0064; ***:p<0.0001).

FIG. 5. Retinoid levels in mouse pancreas following the treatmentindicated. Con fed diet (CFD) (n=5); HFD (n=5). Mice fed a high fatdiet/obese mice have almost no retinoids in the pancreas compared tomice on a control, normal chow diet. They exhibit an organ specificvitamin A deficiency.

FIG. 6. Serum retinol from mice on a high fat diet vs. control dietcompared to the pancreas retinol and retinyl palmitate levels from miceon a high fat vs. control diet. The serum retinol levels are similar ora bit higher in the HF diet mice, but the pancreas retinol levels aremuch lower in the HF diet mice, showing vitamin A deficiency in thepancreas even in the presence of normal serum vitamin A.

FIG. 7. 4-hydroxynonenal (4-HNE), an indicator of oxidative stress, inthe pancreas. The pancreas samples were fixed, embedded in paraffin, andsectioned. Then the tissue sections were stained with an antibodyagainst 4-HNE (magnification, 200×). Sections from two mice/group werephotographed and analyzed. The arrows indicate the pancreatic islets.AC261066 reduces oxidative stress in the pancreatic islets in mice on ahigh fat diet (HFD+AC261066).

FIG. 8. AC261066 slightly reduces expression of c-peptide (marker ofinsulin secretion stress) in islets of HF fed mice. Representativeimmunofluorescence stained pancreatic sections from wild type (wt) maleC57/BL6 mice fed either chow control diet (Con), high fat (HF) diet, HFdiet plus AC261066 for 4 months. Con Diet (n=5); HF diet (n=5); HFDiet+AC261066 (n=5). Blue, nuclei of cells; red, glucagon; green,c-peptide.

FIG. 9. Gene expression of INS2, RARB2, CYP26A1 and LRAT in pancreatictissue from wild type (wt) male C57/BL6 mice fed either chow controldiet (Con), high fat (HF) diet, HF diet plus AC261066. Cyp26 and LRAT,no detectable signal. HPRT, loading control. RAR β2 mRNA levels weredecreased by the high fat diet compared to the control diet (con),consistent with the vitamin A deficiency in the pancreas. AC261066increased the RAR β2 mRNA levels in the HF diet mice.

FIG. 10. Gene expression of RARβ2 in pancreatic tissue from LRAT −/−vitamin A sufficient mice (VAS, normal control diet), LRAT −/− vitamin Adeficient (VAD) mice, and LRAT −/− vitamin A deficient (VAD) micetreated with AC261066 for 8 weeks. AC261066 increased the RARβ2 mRNAlevels in vitamin A deficient mice (LRAT −/− on a VAD diet for 4 months.

FIG. 11. AC261066 diminished hepatic steatosis. Representativehematoxylin and eosin stained liver sections from wild type (wt) maleC57/BL6 mice fed either a chow control diet (Con), high fat (HF) diet,HF diet plus AC261066 or HF diet plus CD1530 (RAR gamma agonist) for 4months. Con Diet (n=5); HF diet (n=5); HF Diet+AC261066 (n=5), or HFdiet+CD1530 (RAR gamma agonist) (n=4).

FIG. 12. Gene expression in livers of control and HF-Fed mice. Geneexpression of SREBP1c and α-SMA in livers from wild type (wt) maleC57/BL6 mice fed either a chow control diet (Con), high fat (HF) diet, aHF diet plus AC261066 or HF diet plus CD1530 (RAR gamma agonist) for 4months. Con Diet (n=5); HF diet (n=5); HF Diet+AC261066 (n=5), or HFdiet+CD1530 (RAR gamma agonist) (n=4).

FIG. 13. AC261066 diminished activation of hepatic stellate cells.Representative immunofluorescence and oil red o stained liver sectionsfrom wild type (wt) male C57/BL6 mice fed either a chow control diet(Con), high fat (HF) diet, HF diet plus AC261066 or HF diet plus CD1530(RAR gamma agonist) for 4 months. Control Diet (n=5); HF diet (n=5); HFDiet+AC261066 (n=5), or HF diet+CD1530 (RAR gamma agonist).

FIG. 14. Gene Expression of Inflammatory Mediators in Livers of LF andHF-Fed Mice. Gene expression of MCP-1, TNF-alpha in livers from wildtype (wt) male C57/BL6 mice fed either a chow control diet (Con), highfat (HF) diet, HF diet plus AC261066 or HF diet plus CD1530 (RAR gammaagonist) for 4 months. LF Diet (n=5); HF diet (n=5); HF Diet+AC261066(n=5), or HF diet+CD1530 (RAR gamma agonist) (n=4). AC261066 decreaseslevels of inflammatory proteins MCP-1 and TNF alpha in livers of HF dietfed mice.

FIG. 15. Mouse serum triglyceride levels following the treatmentsindicated. Con diet (n=2); HFD (n=3); HFDAC (n=5). Con, control diet;HFD, high fat diet; HFD+AC, high fat diet+AC261066. AC261066 does notincrease triglyceride levels at doses used.

FIG. 16. Retinoid levels in mouse liver following the treatmentsindicated. Con fed diet (CFD) (n=5); HFD (n=5); HFD+AC261066 (n=5),HFD+CD1530 (n=4). High fat diet caused a state of vitamin A deficiencyin liver and this is partially reversed by AC261066. Note that they-axes in the left panel are different for CFD and HFD. The HFD reducedretinyl esters, (retinyl palmitate), a form of storage of vitamin A inthe liver, by greater than 90% (left panel). The HFD also reducedretinol (vitamin A) levels by over 90% to result in vitamin A deficiencyin the liver.

FIG. 17. 4-hydroxynonenal (4-HNE), an indicator of oxidative stress, inthe liver. The liver samples were fixed, embedded in paraffin, andsectioned. Then the tissue sections were stained with an antibodyagainst 4-HNE (magnification, 200×). Sections from two mice/group werephotographed and analyzed. These data show that AC261066 reducesoxidative stress and ROS (reactive oxygen species) in the livers of HFdiet fed mice. Oxidative stress damages tissues.

FIG. 18. AC261066 diminished renal lipid accumulation. Representativehematoxylin and eosin stained kidney sections from wild type (wt) maleC57/BL6 mice fed either a chow control diet (Con), high fat (HF) diet,HF diet plus AC261066 or HF diet plus CD1530 (RAR gamma agonist) for 4months. Con Diet (n=5); HF diet (n=5); HF Diet+AC261066 (n=5), or HFdiet+CD1530 (RAR gamma agonist) (n=4).

FIG. 19. AC261066 diminished expression of the fibrogenic proteinalpha-SMA. Representative immunofluorescence and oil red o stainedkidney sections from wild type (wt) male C57/BL6 mice fed either a chowcontrol diet (Con), high fat (HF) diet, or HF diet plus AC261066 for 4months. Chow Diet (n=5); HF diet (n=5); HF Diet+AC261066 (n=5), or HFdiet+CD1530 (RAR gamma agonist).

FIG. 20. Retinoid levels in mouse kidney following the treatmentsindicated. Con fed diet (CFD) (Lean) (n=5) or HFD (Obese)(n=5). The highfat diet led to dramatic declines in retinyl esters (retinyl palmitate)and retinol in the kidney, showing a vitamin A deficiency in kidney.

FIG. 21. Gene Expression of Inflammatory Mediators in Kidneys of ControlNormal Chow (13% fat) and HF- Fed Mice and RARs. Gene expression ofkidney from wild type (wt) male C57/BL6 mice fed either a chow controldiet (Con), high fat (HF) diet, HF diet plus AC261066. AC261066 reducesthe levels of TNF-alpha, a potent inflammatory protein, mRNA in high fatdiet fed mice. AC261066 also restores RAR beta and LRAT mRNA levels,markers of functional vitamin A signaling, in the high fat diet fedmice, 4 months on the HFD. HPRT, loading control.

FIG. 22. 4-hydroxynonenal (4-HNE), an indicator of oxidative stress, inthe kidneys. The kidney samples were fixed, embedded in paraffin, andsectioned. Then the tissue sections were stained with an antibodyagainst 4-HNE (magnification, 200×). Sections from two mice/group werephotographed and analyzed. AC261066 reduces oxidative stress (ROS) inthe kidneys of mice fed the HF diet.

FIG. 23. Retinoid levels in mouse testes following the treatmentsindicated. Con fed diet (CFD) (n=5) or HFD (n=5). High fat diet resultsin partial vitamin A deficiency in the testes.

FIG. 24. Gene expression of vitamin A relevant genes in testes of chowand HF-Fed mice. Gene expression of testes from wild type (wt) maleC57/BL6 mice fed either a chow control diet (Con), high fat (HF) diet,HF diet plus AC261066. (Each number is data from one mouse, five micetotal in each group.)

FIG. 25. AC261066 greatly reduces serum lipids and hepatic lipogenicgene expression. A) Serum triglycerides and cholesterol from controldiet (Con) (n=4); high fat diet (HFD) (n=4); high fat diet (HFD) plusAC261066 (HFD+AC261066 [RARβ agonist]) (n=4), and high fat diet (HFD)plus CD1530 [RARγ agonist])(n=4). B) Hepatic gene expression (mRNA) ofmediators of lipogenesis and gluconeogesis in Con, HFD, HFD+AC261066 andHFD+CD1530-fed mice.

FIG. 26. Modeling structure reproduced from Lund et al., 2005, J. Med.Chem. 48:7517-7519.

FIG. 27. Retinoic Acid Receptor β (RARβ) agonists diminish diet inducedbody weight increases, glucose intolerance and insulin resistance inhigh fat and genetic models of diabetes. A) Body weights of wild typeC57/B16 male mice after 4 months of being fed either: a standard chow(13% Kcal/fat) diet (Wt Con, n=4), a high fat (45% Kcal/fat) diet (HFD,n=5) or Con and HFD with the RARβ agonists AC261066 (Wt Con+AC261, n=3,Wt HFD+AC261, n=4) or AC55649 (Wt Con+AC556, n=3, Wt HFD+AC556, n=4), intheir drinking water. E) Body weights of genetically obese and diabeticOb/Ob and Db/Db mice that were fed either a standard chow diet (n=3 pergroup), or a chow diet plus AC261(n=3 per group) in their drinking wateras described in A for 8 weeks. B-D) and F-H) Fasting glucose, glucosetolerance tests (GTT) and Area Under the Curve Glucose (AUC) from wt andOb/Ob and Db/Db mice described in A an E. I-K) Fasting insulin andinsulin secretion and AUC insulin of Ob/Ob and Db/Db mice subjected toGTT. L-M) Insulin tolerance testing (ITT) of Ob/Ob and Db/Db micedescribed in E. Errors bars represent ±SEM. *=p<0.05,**=p<0.01,***=p<0.001,****=p<0.0001.

FIG. 28. Retinoic Acid Receptor β (RARβ) agonist AC261066 diminishes thenumber of large pancreatic islets and pancreatic insulin content inOb/Ob and Db/Db models of obesity and diabetes. A) Representative imagesof pancreatic islets immunofluorescence stained with antibodies againstinsulin (green) in Wt and Ob/Ob and Db/Db mice fed experimental diets asdescried in FIG. 1A and FIG. 1E respectively. Magnification 400×, ScaleBars=100 μm. B) Relative percentages of very large islets: (area>50,000μm2), large islets: (area=20,000-50,000 μm2) medium islets:(area=5,000-20,000 μm2) and small islets: (area=1,000-5000 μm2) in Wtand Ob/Ob mice fed experimental diets with and without the RARβ agonistas descried in FIG. 27A and FIG. 27E respectively. C) Pancreatic insulincontent (ng/μg of pancreatic protein) in Wt Con and Ob/Ob mice fedexperimental diets with and without the RARβ agonist as descried in FIG.27A and FIG. 27E respectively. Errors bars represent ±SEM. ***=p<0.0001.

FIG. 29. Retinoic Acid Receptor β (RARβ) agonist AC261066 reduces theaccumulation of liver, pancreas, kidney, muscle and adiposetriglycerides in dietary and genetic models of diabetes. A)Representative images of Hematoxylin and Eosin stained liver (a-e),pancreas (f-j), kidney (k-o) and adipose tissue (p-t) from Wt and Ob/Obmice fed experimental diets with and without the RARβ agonist asdescried in FIG. 1A and FIG. 1E. Magnification 200×, Scale Bars=50 μm.B) Percent hepatic steatosis and C-F) tissue triglycerides in Wt andOb/Ob mice fed experimental diets with and without the RARβ agonist asdescried in FIG. 27A and FIG. 27E. G) Total adipose tissue weights in Wtand Ob/Ob mice fed experimental diets with and without RARβ agonist asdescried in FIG. 27A and FIG. 27E. Errors bars represent ±SEM. *=p<0.05,**=p<0.01, ****=p<0.0001.

FIG. 30. Retinoic Acid Receptor β (RARβ) agonist AC261066 alters tissueexpression of genes involved in lipogenesis and mitochondrial oxidationof lipids. Real-time PCR measurements of relative hepatic, pancreaticand adipose tissue transcript levels of genes involved in lipidmetabolism from Wt and Ob/Ob mice fed experimental diets with andwithout the RARβ agonist as descried in FIG. 27A and FIG. 27E. A)Relative hepatic mRNA levels of genes involved in mitochondrialβ-oxidation of lipids. B) Relative hepatic mRNA levels of genes involvedin lipogenesis. C) Relative pancreatic mRNA levels of genes involved inmitochondrial β-oxidation of lipids. D) Relative adipose mRNA levels ofgenes involved in lipid metabolism. Relative fold mRNA levels werenormalized to transcript levels of Hprt. Errors bars represent ±SEM of(n=3-5) animals per experimental group.

FIG. 31. Acute administration of Retinoic Acid Receptor β (RARβ) agonistAC261066 reverses high fat induced glucose intolerance and insulinresistance. A-C) Body weights, water and food intake of wild typeC57/B16 male mice after 3 months of being fed either: a standard chow(13% Kcal/fat) diet (Wt Con, n=4), a high fat (45% Kcal/fat) diet (HFD,n=4) or 3 months of a HFD mice plus 8 days of administration of the RARβagonist AC261066 (n=4) in their drinking water at a dose of 5.4 mg/KgBW/day. On day 8 mice were tested for glucose intolerance and insulinresistance. D and E) Random glucose and glucose tolerance test (GTT) ofmice described in A. F) Insulin tolerance test of mice described in A.

FIG. 32. Retinoic Acid Receptor β (RARβ) agonists diminish diet inducedbody weight increases and glucose intolerance in a high fat model ofdiabetes. A) Body weights of wild type C57/B16 male mice after 3 monthsof being fed either: a standard chow (13% Kcal/fat) diet (Wt Con, n=4),a high fat (45% Kcal/fat) diet (HFD, n=5) or a HFD with the RARβagonists AC261066 (HFD+AC261, n=5) or AC55649 Wt HFD+AC556, n=4), intheir drinking water. B and C) Glucose tolerance tests (GTT) and AreaUnder the Curve Glucose (AUC) from mice described in A. D) Blood glucoselevel in randomly tested (fed) mice and from mice fasted for 16 hours asdescribed in A. Errors bars represent ±SEM. *=p<0.05, **=p<0.01,***=p<0.001.

FIG. 33. Retinoic Acid Receptor β (RARβ) Agonist AC261066 Alters RenalExpression of Genes and Proteins involved in Lipid Metabolism,Inflammation and Fibrosis in Dietary and Genetic Models of Obesity andDiabetes. A) Relative kidney mRNA levels of genes involved inlipogenesis and mitochondrial β-oxidation of lipids from Wt, Ob/Ob andDb/Db mice fed experimental diets with and without the RARβ agonistAC261066. B-D) Relative kidney mRNA levels of genes involved ininflammation (MCP-1 and TNF-α) and fibrosis (α-SMA). E)Semi-quantitative PCR of TNF-α mRNA transcripts. All relative andsemi-quantitative fold mRNA levels were normalized to transcript levelsof Hprt. Errors bars represent ±SEM of (n=3-5) animals per experimentalgroup. F, G) Representative images of kidney tissue sectionsdouble-immunofluorescence stained with antibodies against vimentin(green) and α-SMA in Wt, Db/Db and Db/Db mice given AC261066 in theirwater for 8 weeks. Magnification 400×, Scale Bars=100 μm.

DETAILED DESCRIPTION

As discussed above, there remains a need to provide alternate therapiesor management for the treatment or prevention of certain metabolicsyndrome related conditions, including controlling the level ofcholesterol, triglyceride and/or glucose in a subject in need thereof,as well as treating or preventing diseases caused by fat accumulation orvitamin A deficiency in a subject in need thereof. Accordingly, thepresent invention relates to uses of vitamin A and retinoic acidreceptor β (RARβ) agonists in this regard.

Mouse embryonic stem (ES) cells are pluripotent cells derived from theinner cell mass of blastocyst-stage (day 3.5) embryos (10, 11). Upon LIFremoval, ES cells spontaneously differentiate into all three primaryembryonic germ layers: endoderm, mesoderm, and ectoderm (10). Severalresearch groups have shown that the directed differentiation of ES cellsalong the endocrine pathway can be achieved by using a wide range ofgrowth/differentiation factors, including retinoic acid (RA) treatment(12-17).

Although the effects of RA on cells and tissues are known to occurthrough the activation of retinoic acid receptors (RARα, RARβ, and RARγ)and their isoforms (6, 18), the events occurring downstream of RAsignaling that direct the differentiation of definitive endoderm intoendocrine precursors are poorly understood (4, 5, 19).

A series of in vivo experiments, including some in Xenopus revealed,however, that RA signaling is crucial for endocrine pancreaticdevelopment (20). For instance, mice containing an inducible transgenefor the dominant negative RARα403 mutant, used to ablate retinoicacid-dependent processes in vivo, lacked both a dorsal and ventralpancreas, and died at the neonatal stage (21). Impaired pancreatic isletdevelopment and repletion were also observed in vivo, in vitamin Adeficiency models (22, 23). Moreover, a study of the developmentalpathways involved during in vitro islet neogenesis revealed a 3-foldinduction of RARβ transcripts from “adherent” to “expanded” stages ofendocrine differentiation (24). Another study, based on the role ofCRABP1 and RBP4 in pancreatic differentiation, corroborated theup-regulation of RARβ in early differentiation (11). While previousstudies suggested that RARβ is essential to pancreas development, littleis known about its functional role in pancreas formation and isletmaintenance in adults (25, 26).

Vitamin A metabolite all trans-retinoic acid (RA) acting through itscognate receptors, retinoic acid receptor (RAR) alpha, beta, gamma,possesses anti-obesity and anti-lipogenic properties through regulationof genes involved in energy metabolism and adipogenesis (75).

Using animal models, the present inventors have discovered that retinoicacid receptor β (RARβ) plays an important role in organ development,maintenance, and function. The inventors discovered that vitamin A andRARβ agonists increase RARβ function and signaling; vitamin A and theseRARβ agonists also increase the level of RARβ.

The present inventors also discovered that vitamin A and RARβ agonistsare effective in treating and preventing high fat diet associateddisease in pancreas, liver, kidney, testes, muscle, or adipose tissueand other organs. Furthermore, the inventors discovered that vitamin Aand such (RARβ) agonists can restore vitamin A signaling in organs thatshow vitamin A deficiencies.

Vitamin A and these RARβ agonists, according to the discovery of thepresent inventors, increase insulin signaling, decrease fat deposit,prevent inflammation, and decrease oxidative stress in various organs,including pancreas, liver, kidney, testes, muscle, or adipose tissue.They also decrease the level of alpha smooth muscle actin (α-SMA) butincrease the level of lethicin:retinol acyltransferase (LRAT) and RARβ.When used to treat liver diseases, vitamin A and these RARβ agonistsdecrease the activation of hepatic stellate cells (HSCs) and the levelof hepatic reactive oxygen species (ROS).

The present inventors discovered that vitamin A or agonists of retinoicacid receptor-beta (RARβ) do not elevate serum triglyceride or increasecardiovascular risk at a clinically significant level.

As described above, a common side effect of retinoid agonistadministration to humans and rodents includes both elevated triglycerideand cholesterol levels. The inventors of the present applicationcarefully studied different types of retinoid agonists usingcarefully-designed animal models. Contrary to the reports that retinoidagonists increase triglyceride and cholesterol levels, the inventorsdiscovered that RARβ (e.g., RARβ2) agonists can actually lower serumcholesterol and/or triglyceride level in animals.

The present invention thus also provides pharmaceutical compositionscomprising a RARβ2 agonist, and uses of such agonists to controlhypertriglyceridemia and/or hypercholesterolemia, as well as conditionsassociated thereof.

The retinoic acid receptor (RAR) is a type of nuclear receptor that isactivated by both all-trans retinoic acid and 9-cis retinoic acid. Thereare three retinoic acid receptors (RAR), RARα, RARβ, and RARγ, encodedby the RARα, RARβ, RARγ genes, respectively. Each receptor isoform hasseveral splice variants: two- for α, four- for β, and two- for γ.

RAR heterodimerizes with RXR and in the absence of ligand, the RAR/RXRdimer binds to hormone response elements known as retinoic acid responseelements (RAREs) complexed with corepressor protein. Binding of agonistligands to RAR results in dissociation of corepressor and recruitment ofcoactivator protein that, in turn, promotes transcription of thedownstream target gene into mRNA and eventually protein.

There are three retinoic acid receptors (RAR), RARα, RARβ, and RARγ,encoded by the RARα, RARβ, RARγ genes, respectively. Each receptorisoform has several splice variants: two- for α, four- for β, and two-for γ.

The RARβ subtype consists of four known isoforms RARβ1, RARβ2, RARβ3 andRARβ4. The ligand binding domains of the four isoforms are identical,while the variation between the isoforms is located within the proximalN-terminus, which encompasses the ligand-independent activation domain(AF-1) (Lund et al., 2005, J. Med. Chem. 48:7517-7519).

It has been reported that the ligand binding domain, i.e., AF-2, of agiven RAR isotype cooperates with the AF-1 domain in a promoter contextmanner (Lund et al., 2005, J. Med. Chem. 48:7517-7519; Nagpal et al.,1992, Cell, 70, 1007-1019; Nagpal et al., 1993, EMBO J. 12, 2349-2360.)The AF-2 domains are conserved between the isoforms, the AF-1 domainsare not (Lund et al., 2005, J. Med. Chem. 48:7517-7519, Gelman et al.1999, J. Biol. Chem., 274, 7681-7688; Benecke et al. 2000, EMBO Rep., 1,151-157.) Relying on RARβ (e.g., RARβ2) receptor-ligand crystalstructure, various RARβ agonists have been designed and identified (Lundet al., 2005, J. Med. Chem. 48:7517-7519; Germain et al., 2004, EMBOreports, 5(9): 877-882).

The cooperation and complexes formed between AC261066 and/or AC 55649with AF-1 and AF-2 may serve as an effective model system foridentifying and selecting additional compounds that may be used tocontrol hypertriglyceridemia, hypercholesterolemia and conditionsassociated thereof according to embodiments of the present invention(e.g., FIG. 26).

Known RARβ agonists include but are not limited to: AC261066, AC55649,Tazarotene, Adapalene, 9-cis-retinoic acid, and TTNPB. AC261066 andAC55649 are highly-specific RARβ agonists. The term “highly-specificRARβ agonists” also include other agonists having a binding affinitysimilar to AC261066 or AC55649, e.g., at least 50% or greater,preferably 75% or greater, more preferably 90% or greater of the RARβbinding affinity of AC261066 or AC55649. The term “highly-specific RARβ2agonists” include agonists having a binding affinity similar to AC261066or AC55649, e.g., at least 50% or greater, preferably 75% or greater,more preferably 90% or greater of the RARβ2 binding affinity of AC261066or AC55649. A highly-specific RARβ (e.g., RARβ2) agonist preferably hasan affinity for RARβ (e.g., RARβ2) greater than 6.00 pEC50, morepreferably greater than 6.50 pEC50, more preferably greater than 7.00pEC50, more preferably greater than 7.50 pEC50, more preferably greaterthan 7.75 pEC50, and even more preferably greater than 8.00 pEC50.

RARβ agonists include the fluorinated alkoxythiazoles previouslydescribed (65), such as:

4′-Octyl-[1,1′-biphenyl]-4-carboxylic acid (65), Adapalene (67),BMS-231973, BMS-228987, BMS-276393, BMS-209641 (66), BMS-189453{4-[(1E)-2-(5,6-Dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]-benzoicacid} (68), CD2019(6-[4-methoxy-3-(1-methylcyclohexyl)phenyl]naphthalene-2-carboxylicacid), compounds described in WO2008/064136 and WO2007009083 andtazarotene (ethyl6-[2-(4,4-dimethyl-3,4-dihydro-2H-1-benzothiopyran-6-yl)ethynyl]pyridine-3-carboxylate). Structures of some RARβ agonists are providedbelow:

RARβ agonists also include those disclosed in published PCT patentapplication WO2008/064136, WO2007/009083 and published U.S. patentapplication US2009/0176837, each of which is incorporated herein byreference in its entirety. The highly specific RARβ agonists, e.g.,AC261066 and AC55649, are highly isoform-selective agonists for thehuman RARβ2 receptors as described in Lund et al. (2005, J. Med. Chem.,48, 7517-7519, incorporated herein by reference in its entirety).

RARβ2 receptor agonist of the present invention may be selected from thefollowing compounds or an ester thereof (RARβ2 binding activitiesindicated in Tables 1 and 2 below):

The functional receptor assay, receptor selection and amplification maybe performed as described in WO2007/009083. For example, Technology(R-SAT) may be used to investigate the pharmacological properties ofknown and novel RARβ agonists useful for the present invention. R-SAT isdisclosed, for example, in U.S. Pat. Nos. 5,707,798, 5,912,132, and5,955,281, Piu et al., 2006, Beta Arrestin 2 modulates the activity ofNuclear Receptor RAR beta 2 through activation of ERK2 kinase, Oncogen,25(2):218-29 and Burstein et al., 2006, Integrative Functional Assays,Chemical Genomics and High Throughput Screening: Harnessing signaltransduction pathways to a common HTS readout, Curr Pharm Des, 12(14):1717-29 all of which are hereby incorporated herein by reference intheir entireties, including any drawings.

The relevant RARβ2 receptor modulating activities of the above compoundsare provided in Table 1 and Table 2 of WO2007/009083:

TABLE 1 RARβ2 Compound no. % Eff. pEC50 1 39 8.64 2 126 8.10 3 107 8.024 44 7.82 6 104 7.73 8 79 7.66 9 76 7.59 10 64 7.58 11 78 7.56 12 727.54 13 85 7.38 15 76 7.37 16 37 7.34 18 108 7.32 20 98 7.26 22 36 7.2424 58 7.21 25 65 7.20 26 36 7.15 27 95 7.12 29 78 7.08 31 80 7.02 32 707.02 33 98 6.96 35 83 6.94 36 42 6.91 37 49 6.87 38 70 6.81 44 41 6.6145 78 6.59 46 54 6.58 47 58 6.58 49 59 6.55 50 85 6.53 51 59 6.51 52 456.41 53 99 6.29 54 39 6.18 55 84 6.17 56 105 6.17 57 77 6.17 58 66 6.1559 35 6.11 60 51 6.08 61 39 6.08 62 37 6.08 63 36 6.05 64 77 6.00

TABLE 2 RARβ2 Compound no. % Eff. pEC50 5 124 7.79 7 95 7.71 14 93 7.3817 33 7.34 19 36 7.28 21 106 7.26 23 46 7.22 28 70 7.09 30 62 7.04 34 716.95 39 42 6.76 40 82 6.75 41 25 6.75 42 46 6.71 43 67 6.67 48 49 6.56RARβ2 Compound no. % Eff. pEC50 5 124 7.79 7 95 7.71 14 93 7.38 17 337.34 19 36 7.28 21 106 7.26 23 46 7.22 28 70 7.09 30 62 7.04 34 71 6.9539 42 6.76 40 82 6.75 41 25 6.75 42 46 6.71 43 67 6.67 48 49 6.56

The highly specific RARβ agonist, e.g., AC261066, can prevent hepaticsteatosis and activation of HSCs, marked by decreased expression ofα-SMA. AC261066 can significantly diminish hepatic gene expression ofpro-inflammatory mediators tumor necrosis factor-alpha (TNFα) andmonocyte chemotactic protein-1 (MCP-1).

As used herein, the term “subject” means an animal, preferably a mammal,and most preferably a human. A subject in need thereof may be a patienthaving a metabolic syndrome related condition as discussed herein. Forexample, the subject may have insulin resistance, hypertension (highblood pressure), vitamin A deficiency, diabetes, fatty liver, high bloodpressure, insulin resistance, obesity, abnormal (e.g., elevated)cholesterol, triglyceride and/or glucose levels, artery and heartdiseases. Vitamin A deficiency and abnormal (e.g., elevated)cholesterol, triglyceride and/or glucose levels may be indicated bymeasurement in serum or a non-serum sample, including a sample from anorgan (e.g., pancreas, liver, kidney, testes, muscle, or adiposetissue), of an animal, e.g., human.

As used herein, the term “vitamin A deficiency” refers to a lack ofvitamin A or related metabolites including trans retinol, or a decreasedlevel thereof in a serum sample or a non-serum sample, including asample from an organ (e.g., pancreas, liver, kidney, testes, muscle, oradipose tissue), of an animal, e.g., human. An animal having vitamin Adeficiency according to the present invention may have a normal vitaminA (or related metabolites) level as measured using a serum sample, butstill exhibits vitamin A deficiency as measured using a non-serum samplefrom the animal.

As used herein, the term “hyperglycemia” refers to a condition of highblood sugar in which an excessive amount of glucose circulates in theblood plasma. This is generally a glucose level higher than 11.1 mmol/l(200 mg/dl), but symptoms may not start to become noticeable until evenhigher values such as 15-20 mmol/l (˜250-300 mg/dl). A subject with aconsistent range between ˜5.6 and ˜7 mmol/l (100-126 mg/dl) (AmericanDiabetes Association guidelines) is considered hyperglycemic, whileabove 7 mmol/l (126 mg/dl) is generally held to have diabetes. Chroniclevels exceeding 7 mmol/l (125 mg/dl) can produce organ damage.

As used herein, the term “hypertriglyceridemia” refers to a condition inwhich the triglyceride level is elevated with regard to the normalaverage level of triglycerides in a respective reference subjecttypically of the same ethnic background, age and gender. Typically,triglyceride tests are blood tests that measure the total amount oftriglycerides in the blood. The National Cholesterol Education Program(NCEP) sets guidelines for fasting triglyceride levels as follows:normal triglycerides means there are less than 150 milligrams perdeciliter (mg/dL); borderline high triglycerides are 150 to 199 mg/dL;while high triglycerides are 200 to 499 mg/dL and very hightriglycerides are 500 mg/dL or higher (NCEP, Expert Panel on Detection,Evaluation, and Treatment of High Blood Cholesterol in Adults (AdultTreatment Panel III), 2002, Circulation, 106:3143-3421). By way of anon-limiting example, with regard to humans the termhypertriglyceridemia in particular refers to blood triglyceride levelsabove about 150 mg/dl, in particular above about 180 mg/dl, or it couldbe a lower level that a physician treating the subject would consider tobe significant. For borderline patients, non-pharmacologic measures areusually prescribed, e.g., a change in lifestyle including increasedexercise, low fat diet and smoking cessation. When levels oftriglycerides are greater than 200 mg/dL, drug treatment is typicallygiven.

As used herein, the term “hypercholesterolemia” refers to a condition inwhich the cholesterol level is elevated with regard to the normalaverage level of cholesterol in a respective reference subject typicallyof the same ethnic background, age and gender. Typically, cholesteroltests are blood tests that measure the total amount of cholesterol inthe blood. By way of non-limiting example, with regard to humans theterm in particular refers to blood cholesterol levels above about 200,in particular above about 240 mg/dl, or it could be a lower level that aphysician treating the subject would consider to be significant.

Often, tests for cholesterol are done with fasting for 9 to 12 hours,and it provides results for four different types of lipids (lipidpanels).

Total cholesterol

LDL (low-density lipoprotein), the “bad cholesterol”

HDL (high-density lipoprotein), the “good cholesterol”

Triglycerides, another form of fat in the blood

Some lipid panels provide more detailed information, with information onthe presence and sizes of various fat particles in the blood.

As a non-limiting guideline, for total cholesterol: 200 milligrams perdeciliter (mg/dL) or less is considered normal. 201 to 240 mg/dL isborderline. Greater than 240 mg/dL is considered high.

As a non-limiting guideline, for HDL (“good cholesterol”), more isbetter: HDL 60 mg/dL or higher is good—it protects against heartdisease. HDL between 40 and 59 mg/dL are acceptable. Less than 40 mg/dLHDL is low, increasing the risk of heart disease.

As a non-limiting guideline, for LDL (“bad cholesterol”), lower isbetter: An LDL of less than 100 mg/dL is optimal. An LDL of 100 to 129mg/dL is near-optimal. LDL between 130 and 159 mg/dL is borderline high.LDL cholesterol between 160 and 189 mg/dL is high. An LDL of 190 mg/dLor more is considered very high.

As used herein, the term “condition associated withhypertriglyceridemia” refers to a disease condition which can be causedby and have as a symptom of elevated blood triglyceride levels, or acondition in which a physician treating the subject would considercontrolling the level of triglyceride as helpful for the treatment orprevention of the condition. Conditions associated withhypertriglyceridemia include, but are not limited to, hyperlipidemia,atherosclerosis, cardiovascular diseases, stroke, insulin resistance,diabetes mellitus, diabetic nephropathy, idiopathic pancreatitis,metabolic syndrome, high blood pressure, obesity, high sugar diet,alcohol abuse, chronic renal failure, Rett Syndrome, and glycogenstorage diseases, etc. Conditions associated with hypertriglyceridemiaalso include situations where the treatment of an unrelated diseasecauses elevated triglyceride levels as a drug side effect.

Rett Syndrome is an X-linked syndrome characterized by a series ofsymptoms, including loss of language, loss of coordination, and anautism-like presentation. Some of the effects of this syndrome appear toresult from dysregulation of both cholesterol and triglyceridemetabolism and might be treated with statins (Buchovecky et al, NatureGenetics, 45(9):1013-20; Justice, M J, Seminar at Sloan KetteringInstitute, May 1, 2014).

Hypertriglyceridemia may be classified as either primary or acquired(Assmann, et al., 1991, Am. J. Cardiol., 68: 13A-16A; Mancini et al.,1991, Am. J. Cardiol., 68: 17A-21A). Primary hypertriglyceridemias areinherited disorders, which include chylomicronemia (type Ihyperlipoproteinemia), type V hyperlipoproteinemia, type IIIhyperlipoproteinemia (remnant hyperlipidemia or familialdysbetalipoproteinemia), familial hypertriglyceridemia, familialcombined hyperlipidemia, and hepatic lipase deficiency (Assmann et al.,1991). The severity of the symptoms depends in part on whether thepatient is homozygous or heterozygous. Primary hypertriglyceridemias maypresent as early as childhood. Acquired hypertriglyceridemia may beattributed to many factors, including metabolic disorders such as typeII diabetes, diabetic nephropathy, metabolic syndrome, insulinresistance, pre-diabetes, syndrome X, obesity, hyperuricemia, Alstrom'ssyndrome, Rett Syndrome, and type I glycogen storage disease (Kreisberg,1998, Am. J. Cardiol., 82: 67U-73U; Schmidt et al., 1996, Metabolism ,45: 699-706; Paisey et al., 2004, Clin. Endocrinol., 60: 228-231; Greeneet al., 1991, J Pediatr., 119: 398-403). These conditions may alsopresent in childhood. Similarly, hormonal disturbances may causehypertriglyceridemia. In addition to insulin, triglyceride levels may beelevated as a result of hypothyroidism or polycystic ovary syndrome(Kvetny et al., 2004, Clin. Endocrinol., 61: 232-238; Pirwany et al.,2001, Clin. Endocrinol., 54: 447-453).

Acquired hypertriglyceridemia can be due to lifestyle factors such asdiet (high sugar or carbohydrate intake) or alcohol consumption(Coughlan et al., 2000, Postgrad. Med., 108: 77-84). Chronic diseasestates such as renal disease (including nephrotic syndrome and renalfailure) or paraproteinemia can also cause elevated triglycerides(Attman et al., 1997, Contrib. Nephrol., 120:1-10; Oda et al., 1998,Nephrol. Dial. Transplant., 13:45-49; Matteucci et al., 1996, Clin.Rheumatol., 15:20-24.). These disorders may also manifest in childhood.

As used herein, the term “condition associated withhypercholesterolemia” refers to a disease condition which can be causedby and have as a symptom of elevated blood cholesterol levels, or acondition in which a physician treating the subject would consider ithelpful for the treatment or prevention of the condition by controllingthe level of cholesterol of the subject. Conditions associated withhypercholesterolemia include, but are not limited to, hyperlipidemia,atherosclerosis, cardiovascular diseases, myocardial infarction, stroke,angina pectoris, ischemic colitis, transient ischemic attacks, RettSyndrome, and peripheral artery disease. Conditions associated withhypercholesterolemia also include situations where the treatment of anunrelated disease causes elevated cholesterol level as a drug sideeffect.

Causes for conditions associated with hypercholesterolemia can be forexample diabetes, diabetic nephropathy, nephritic syndrome, overweight,gout, alcohol abuse, hypothyroidism, anorexia nervosa, Zieve's syndrome,pregnancy, Rett Syndrome, and metabolic syndrome. There is also agenetic form of this metabolic derangement resulting in familialhypercholesterolemia. In addition, hypercholesterolemia contributesdirectly to the pathology of many forms of disease conditions, e.g.atherosclerosis, cardiovascular diseases, myocardial infarction, stroke,angina pectoris, ischemic colitis, transient ischemic attacks, and/orperipheral artery disease.

Some types of hypercholesterolemia lead to specific physical findings.For example, familial hypercholesterolemia (Type IIahyperlipoproteinemia) may be associated with xanthelasma palpebrarum(yellowish patches underneath the skin around the eyelids), arcussenilis (white or gray discoloration of the peripheral cornea), andxanthomata (deposition of yellowish cholesterol-rich material) of thetendons, especially of the fingers. Type III hyperlipidemia may beassociated with xanthomata of the palms, knees and elbows.

Longstanding elevation of serum cholesterol can lead to atherosclerosis.Over a period of decades, chronically elevated serum cholesterolcontributes to formation of atheromatous plaques in the arteries. Thiscan lead to progressive stenosis (narrowing) or even complete occlusion(blockage) of the involved arteries. Alternatively smaller plaques mayrupture and cause a clot to form and obstruct blood flow. A suddenocclusion of a coronary artery results in a myocardial infarction orheart attack. An occlusion of an artery supplying the brain can cause astroke. If the development of the stenosis or occlusion is gradual,blood supply to the tissues and organs slowly diminishes until organfunction becomes impaired. At this point, tissue ischemia (restrictionin blood supply) may manifest as specific symptoms. For example,temporary ischemia of the brain (commonly referred to as a transientischemic attack) may manifest as temporary loss of vision, dizziness andimpairment of balance, aphasia (difficulty speaking), paresis (weakness)and paresthesia (numbness or tingling), usually on one side of the body.Insufficient blood supply to the heart may manifest as chest pain, andischemia of the eye may manifest as transient visual loss in one eye.Insufficient blood supply to the legs may manifest as calf pain whenwalking, while in the intestines it may present as abdominal pain aftereating a meal.

Hyperlipidemias are conditions of abnormal plasma lipid/lipoproteinlevels. Specific types of hyperlipidemias associated with vasculardisease include Type IIb and Type IV hyperlipidemias. Type IVhyperlipidemia is characterized by elevated plasma levels of very lowdensity lipoprotein (VLDL). Type IIb hyperlipidemia is characterized byelevated levels of VLDL and low density lipoprotein (LDL). One of themajor causes of atherosclerosis and the related diseases, coronary heartdisease (CHD), peripheral arterial disease (PAD), and cerebrovasculardisease, is dyslipidemia. Dyslipidemia is an imbalance of each of thelipid components: total cholesterol (TC), high density lipoprotein (HDL)cholesterol, low density lipoprotein (LDL) cholesterol, and serumtriglycerides. Because of their link with vascular disease, a number ofapproaches for controlling hyperlipidemias have been developed. Suchapproaches include changes in lifestyle, e.g. diet, exercise, and thelike, as well drug therapy. Drugs finding use in the management ofplasma lipid profiles include: bile acid binding resins; niacin; HMG-CoAreductase inhibitors; fibric acid derivatives, e.g. gemfibrozil; and thelike.Lipoproteins are classified by their density: very low densitylipoprotein (VLDL), intermediate density lipoprotein (IDL), low densitylipoprotein (LDL) and high density lipoprotein (HDL). All thelipoproteins carry cholesterol, but elevated levels of the lipoproteinsother than HDL (termed non-HDL cholesterol), particularlyLDL-cholesterol, are associated with an increased risk ofatherosclerosis and coronary heart disease. In contrast, HDL (“good”cholesterol) helps remove cholesterol from the body tissues by effluxand carry cholesterol back to the liver for disposal. A high level ofHDL cholesterol may lower one's chances of developing heart disease orstroke. LDL (“bad” cholesterol) carries mostly fat and only a smallamount of protein from the liver to other parts of the body. A certainlevel of LDL in the blood is normal and healthy because LDL movescholesterol to the parts of the body that need it. But it is sometimescalled “bad cholesterol” because a high level may increase one's chancesof developing heart disease. VLDL contains very little protein and itdistributes the triglyceride produced by the liver. A high VLDLcholesterol level (e.g., as in hypercholesterolemia) can cause thebuildup of cholesterol in the arteries and increases the risk of heartdisease and stroke. An increase in plasma triglyceride levels causes adecrease in HDL levels.

Elevated levels of non-HDL cholesterol and LDL (“bad” cholesterol) inthe blood may be a consequence of diet, obesity, inherited (genetic)diseases (such as LDL receptor mutations in familialhypercholesterolemia), or the presence of other diseases such asdiabetes and an underactive thyroid.

The inventors of the present application have discovered and identifiedthat specific RARβ (e.g., RARβ2) agonists significantly decreasecirculating triglycerides and cholesterol levels. The present inventionthus establishes a new, specific role for RARβ (e.g., RARβ2) agonists inlowering cholesterol and triglycerides in animals. The present inventionprovides pharmaceutical compositions comprising a RARβ (e.g., RARβ2)agonist, and uses of such RARβ (e.g., RARβ2) agonists for controllingthe levels of triglyceride and/or cholesterol in a subject in needthereof. In addition, the inventors discovered that specific RARβ (e.g.,RARβ2) agonists reduce the production of HMG-CoA reductase andtranscription factor, e.g., by reducing HMG-CoA reductase mRNA orprotein levels. The specific RARβ (e.g., RARβ2) agonists of the presentinvention also reduce the production (e.g., at mRNA and protein levels)of the sterol regulatory element binding protein 2 (SREBP-2), atranscription factor that regulates the genes of cholesterol metabolism.SREBP-2 increases HMG-CoA reductase mRNA.

HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase or HMGCR)is the rate-controlling enzyme of the mevalonate pathway which leads tothe production of cholesterol, isoprenoids and related molecules.HMG-CoA reductase is the target of statins, a collection of drugs thatare prescribed to limit cholesterol production, thus treating heartdiseases. The reduction of HMG-CoA reductase production could lead to adecrease in the amount of cholesterol production.

As used herein, the term “control” refers to decrease, reduce ormaintain the level of a molecule, e.g., triglyceride, cholesterol orglucose in a subject. The level may be measured in a serum sample or anon-serum sample, including a sample from an organ (e.g., pancreas,liver, kidney, testes, muscle, or adipose tissue). The control may be atthe stage of triglyceride or cholesterol synthesis, transport orfunction. As used herein, the terms “decrease” and “reduce” are usedinterchangeably to refer to a negative change in the level, activity orfunction of a molecule, cell or organ. It is meant that the particularlevel, activity or function is lower by about 25%, about 50%, about 75%,about 90%, about 1-fold, about 2-fold, about 5 fold, about 10-fold,about 25-fold, about 50-fold, or about 100 fold, or lower, when comparedto a control.

As used herein, the terms “elevate”, “increase”, “improve” and “enhance”are used interchangeably to refer to a positive change in the level,activity or function of a molecule, cell or organ. It is meant that theparticular level, activity or function is higher by about 25%, about50%, about 75%, about 90%, about 1-fold, about 2-fold, about 5 fold,about 10-fold, about 25-fold, about 50-fold, or about 100 fold, orhigher, when compared to a control.

The expression “therapeutically effective” or “therapeutic effect”refers to a benefit including, but not limited to, the treatment oramelioration of symptoms of a condition discussed herein. It will beappreciated that the therapeutically effective amount or the amount ofagent required to provide a therapeutic effect will vary depending uponthe intended application (in vitro or in vivo), or the subject anddisease condition being treated (e.g., nature of the severity of thecondition to be treated, the particular inhibitor, the route ofadministration and the age, weight, general health, and response of theindividual patient), which can be readily determined by a person ofskill in the art. For example, an amount of vitamin A or an agonist ofRARβ is therapeutically effective if it is sufficient to effect thetreatment or amelioration of symptoms of a condition discussed herein.

The term “clinically significant side effect” is used herein to refer toa level of an undesired side effect caused by the administration of anydrug or pharmaceutical composition that a physician treating the subjectwould consider significant. Such side effect may be elevatedtriglyceride and/or cholesterol level (e.g., in a serum sample or anon-serum sample, including a sample from an organ (e.g., pancreas,liver, kidney, testes, muscle, or adipose tissue)) or an increasedcardiovascular risk. The term “clinically significant level” is usedherein to refer to a level of a side effect such as cardiovascular riskcaused by the administration of a pharmaceutical composition (e.g.,vitamin A or RARβ agonist) that a physician treating the subject wouldconsider to be significant.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 30%, preferably 20%, more preferably 10%.

As used herein, the term “comprises” means “includes, but is not limitedto.”

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio.

If a pharmaceutically acceptable salt of vitamin A or agonist of RARβ isutilized in pharmaceutical compositions, the salt preferably is derivedfrom an inorganic or organic acid or base. For reviews of suitablesalts, see, e.g., Berge et al, J. Pharm. Sci. 66: 1-19 (1977) wadRemington: The Science and Practice of Pharmacy, 20th Ed., ed. A.Gennaro, Lippincott Williams & Wilkins, 2000.

The term “pharmaceutically acceptable carrier” is used herein to referto a material that is compatible with a recipient subject, preferably amammal, more preferably a human, and is suitable for delivering anactive agent to the target site without terminating the activity of theagent. The toxicity or adverse effects, if any, associated with thecarrier preferably are commensurate with a reasonable risk/benefit ratiofor the intended use of the active agent.

The term “carrier” is used interchangeably herein, and includes any andall solvents, diluents, and other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington: TheScience and Practice of Pharmacy, 20th Ed. , ed. A. Gennaro, LippincottWilliams & Wilkins, 2000 discloses various carriers used in formulatingpharmaceutically acceptable compositions and known techniques for thepreparation thereof.

The pharmaceutical compositions of the invention can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders, tablets,capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions. Formulations may optionally contain solvents,diluents, and other liquid vehicles, dispersion or suspension aids,surface active agents, pH modifiers, isotonic agents, thickening oremulsifying agents, stabilizers and preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired.

The vitamin A or agonist of RARβ can be administered by any method knownto one skilled in the art. For example, vitamin A or agonist of RARβ maybe administered orally or parenterally.

The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra- articular, intra- synovial, infrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally, intravenously, or subcutaneously. The formulations of theinvention may be designed to be short-acting, fast-releasing, orlong-acting. Still further, compounds can be administered in a localrather than systemic means, such as administration (e.g., by injection)at a tumor site.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents such as phosphates orcarbonates.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart.

Combination therapies that comprise the combination of vitamin A andagonist of RARβ of the present invention, and further with one or moreother therapeutic agents can be used, for example, to: 1) enhance thetherapeutic effect(s) of the methods of the present invention and/or theone or more other therapeutic agents; 2) reduce the side effectsexhibited by the methods of the present invention and/or the one or moreother therapeutic agents; and/or 3) reduce the effective dose of vitaminA or agonist of RARβ of the present invention and/or the one or moreother therapeutic agents.

The RARβ (e.g., RARβ2) agonist of the present invention may be used incombination with a second drug for treating hypertriglyceridemia andhyertryglyceridemia associated condition. Non-limiting examples of thesecond drug may be a fat absorption inhibitors by blocking pancreatictriglyceride lipase in the intestine, such as Orlistat; a thermogenicagent which increases basal metabolism rate and “fat burning”, such asthyroid hormones and β3-adrenergic agonists; or an appetite suppressantdrug (suppression of food intake), such as serotonin agonists,sympathomimetic agents and leptin.

The RARβ (e.g., RARβ2) agonist of the present invention may be used incombination with a second drug for treating hypercholesterolemia andhypercholesterolemia-associated condition. Non-limiting examples of thesecond drug may be an HMG-CoA reductase inhibitor (inhibitors forcholesterol biosynthesis; so-called “statins”); a cholesterol absorptioninhibitor (such as ezetimibe); a bile acid sequestrant (such ascholestyramine and colestipol); a fibric acid derivative (such asfenofibrate and gemfibrozil); a high dose (3-6 g/day) of niacin; anapolipoprotein inhibitor (such as monoclonal antibodies and antisenseoligonucleotides mipomersen and ISIS-APOCIII); a proprotein convertasesubtilisin/kexin type 9 (PCSK9) inhibitor (such as monoclonal antibodiesalirocumab, evolocumab, bococizumab, RG-7652 and LY3015014, as well asantisense oligonucleotides ALN-PCS02 and SPC5001).

In a particular embodiment of the invention the pharmaceuticalcomposition for use in the present invention is also compatible withphysical exercise, a diet and/or dietary therapy approaches to reduce orprevent hypertriglyceridemia and/or hypercholesterolemia. Such diets ordietary therapies relate e.g. to the reduction of caloric intake, fatintake and/or cholesterol intake. In addition, reducing saturateddietary fat may be recommended to reduce total blood cholesterol and LDLin a subject in need thereof according to the present invention. Ifnecessary, other treatments such as LDL apheresis or even surgery (forparticularly severe subtypes of familial hypercholesterolemia) may beperformed.

Many drugs available to treat diseases have been reported to elevatetriglyceride or cholesterol levels in patents. The RARβ (e.g., RARβ2)agonist of the present invention may be used in combination with asecond drug used to treat an unrelated condition where the second drugcauses a clinically significant level of side effect by increasingtriglyceride and/or cholesterol levels in the subject. The second drugincludes but is not limited to one selected from the group consisting ofdiuretics (including thiazide) and loop diuretics; β-blockers (e.g.,Atenolol, Bisoprolol, Metoprolol, Nadolol, Propanolol); proteaseInhibitors; angiotensin converting enzyme inhibitors; estrogenreplacement therapy; oral contraceptives with second and thirdgeneration progestogens; estrogen receptor modulators; prednisones,amiodarones; cyclosporine; progestin; anabolic steroids; retinoids; andacitretin; immunosuppressive drugs (such as rapamycin); proteaseinhibitors (such as ritonavir); indinavir; and nelfinavir; andantipsychotics (such as clozapine).

The amount or suitable dosage of vitamin A or agonist of RARβ dependsupon a number of factors, including the nature of the severity of thecondition to be treated, the route of administration and the age,weight, general health, and response of the individual subject. Incertain embodiments, the suitable dose level is one that achieves thistherapeutic response and also minimizes any side effects associated withthe administration. For example, vitamin A or agonist of RARβ may beadministered at an amount from about 30 mg to about 200 mg per day,e.g., about 50 mg to about 150 mg per day, about 50 to about 100 mg perday, about 100 mg to about 150 mg per day.

Vitamin A or agonist of RARβ may be administered in single or divided ormultiple doses. It will be understood that a suitable dosage of vitaminA or agonist of RARβ may be taken at any time of the day or night, withfood or without food. In some embodiments, the treatment period duringwhich an agent is administered is then followed by a non-treatmentperiod of a particular time duration, during which the therapeuticagents are not administered to the patient. This non-treatment periodcan then be followed by a series of subsequent treatment andnon-treatment periods of the same or different frequencies for the sameor different lengths of time. The expression profile of one or more suchgenes (e.g., as listed in Table 5 below) may be a therapeutic effectindicator which may be used to direct therapeutic regimen and dosesaccording to the present invention.

The therapeutic effect of vitamin A or retinoic acid receptor-beta(RARβ) agonist may be achieved relatively quickly from about 1 day toabout 8 days (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days)after it is administered to the subject in need, it may take longertime.

The pharmaceutical composition for use in the present invention can beadministered when triglyceride, cholesterol and/or glucose levels arealready elevated in a subject, but can also be administered in advance,if the triglyceride, cholesterol and/or glucose levels are expected torise in the near future or if any potential rise of triglyceride,cholesterol or glucose levels would be detrimental for the health and/orstatus of the patient. In the latter case (detrimental effect) thepharmaceutical composition for use in the present invention is inparticular administered to anticipate and prevent peaks of triglyceride,cholesterol and/or glucose levels.

In the following description, reference is made to the accompanyingdrawings that form a part hereof, and in which is shown by way ofillustration specific embodiments which may be practiced. Theseembodiments are described in detail to enable those skilled in the artto practice the invention, and it is to be understood that otherembodiments may be utilized and that logical changes may be made withoutdeparting from the scope of the present invention. The followingdescription of example embodiments is, therefore, not to be taken in alimited sense, and the scope of the present invention is defined by theappended claims.

EXAMPLES

The present description is further illustrated by the followingexamples, which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication) are hereby expressly incorporated by reference.

Example 1—Materials and Methods

Cell Culture and Isolation of RARβ Homozygous ES Cell Line. Mouse J1wild-type ES cells were cultured as described previously (27). C57BL/6RARβ heterozygous mice were provided by Dr. Pierre Chambon(Strasbourg-Cedex, France) (26). Homozygous RARβ-null mice were obtainedfollowing mating of RARβ heterozygous mice. Blastocysts were harvestedon day E3.5 and individually cultured on ES cell medium as previouslydescribed (28).

Pancreatic endocrine differentiation protocol. A slightly modifiedversion of the established protocols published by Borowiak (14) andD'Amour (15) was used to carry out differentiation of hormone expressingendocrine cells from mouse ESCs. Prior to differentiation, ESCs wereseeded at 5×10⁵ on 30 mm gelatin-coated plates. After overnight culture,cells were exposed to 250 nM BIO-Acetoxime (EMD Bioscience, San Diego,Calif.) +50 ng/ml activin A (R&D Systems, Minneapolis, Minn.) inAdvanced RPMI (GIBCO, Grand Island, N.Y.) supplemented with 1× L-Glu and0.2% FBS (GIBCO) for 1 day, and then to activin A alone in the samemedia. Cells were then cultured for 4 days to induce endodermdifferentiation. For pancreatic progenitor induction, the cells weretransferred to 50 ng/ml FGF10 (R&D Systems), 7.5 μM cyclopamine(Calbiochem, San Diego, Calif.) in DMEM supplemented with 1× L-Glu, 1×Pen/Strep, and 1× B27 (Invitrogen, Grand Island, N.Y.) for 2 days. Atday 7, cells were transferred to FGF10, cyclopamine and 2 μM all-transRA (Sigma, St. Louis, Mo.) in DMEM supplemented with 1× L-Glu, 1×Pen/Strep, and 1× B27 (Invitrogen) for 4 days. At day 11, cells werecultured in the presence of DMEM supplemented with 1× L-Glu, 1×Pen/Strep, and 1× B27 for 3 days. At day 14, CMRL (Invitrogen) mediumwas added and supplemented with 1× L-Glu, 1× Pen/Strep, 1× B27, 50 ng/mlIGF-1 (R&D Systems), 50 ng/ml HGF (R&D Systems), and 10 mM nicotinamide(Sigma) for 3 more days. All stock compounds were made in either PBS orethanol.

RT-PCR analysis. Various markers for endodermal (day 5), pancreaticprogenitor (day 11), endocrine progenitor (day 14) and endocrine (day17) differentiation were analyzed by semi-quantitative RT-PCR in J1wild-type and RARβ KO ESCs. Specific primers used and amplificationconditions are listed in Table-3. Primers were designed around intronswhenever possible. Primers not designed around introns are shown inTable 1 with an asterisk. Total RNA extraction, semi-quantitative andquantitative PCR reactions were performed as previously described (18).Amplified PCR products were resolved on 1.5% agarose gels and visualizedby staining with ethidium bromide. PCR bands were sequenced forverification of the correct amplicon. Quantitation of semi-quantitativegels was performed using ImageJ software (National Institutes of Health)from three experimental biological repeats.

TABLE 3 Primer sequences used for RT-PCRAll primers for RT-PCR are designed aroundintrons, except those marked with *. Forward Reverse Product Applica-Sequence Sequence size Primer tion (5′→3′) (5′→3′) (bp) mIns1 RT-PCRTAGTGACCAG ACGCCAAGG 289 CTATAATCAG TCTGAAGGT AG CC (SEQ ID (SEQ IDNo. 1) No. 2) mGcg RT-PCR CCGCCGTGCC CCTGCGGCC 232 CAAGATTTT GAGTTCCT(SEQ ID (SEQ ID No. 3) No. 4) mSst* RT-PCR GAGCCCAACC GAAGTTCTTG 150AGACAGAGA CAGCCAGCTT A(SEQ ID (SEQ ID No. 5) No.6) mNgn3* RT-PCRCTGCGCATAG CTTCACAAGA 233 CGGACCACAG AGTCTGAGAA CTTC CACCAG (SEQ ID(SEQ ID No. 7) No. 8) MRaRβ RT-PCR GATCCTGGAT CACTGACGCC 248 TTCTACACCGATAGTGGTA (SEQ ID (SEQ ID No. 9) No.10) mNanog RT-PCR AAAGGATGAACTGGCTTTG 520 GTGCAAGCGG CCCTGACTT TGG TAA (SEQ ID (SEQ ID No. 11)No. 12) mRex1 RT-PCR GAAAGCAGGA CGATAAGACAC 641 TCGCCTCACTG CACAGTACACTGC AC (SEQ ID (SEQ ID No. 13) No. 14) mCyp26a1 RT-PCR GAAACATTGCACGGCTGAAGG 272 GATGGTGCTTC CCTGCATAAT AG CAC (SEQ ID (SEQ ID No. 15)No. 16) mPax-6 RT-PCR GCAACCCCCAG AGTCCATTCC 399 TCCCCAGTCAG CGGGCTCCAGA TTCA (SEQ ID (SEQ ID No. 17) No. 18) mIsl-1* RT-PCR CCCGGGGGCCACGGGCACGC 397 CTATTTG ATCACGAA (SEQ ID (SEQ ID No. 19) No. 20) mIapp*RT-PCR TGGGCTGTAGT GCACTTCCGT 199 TCCTGAAGC TTGTCCATCT (SEQ ID (SEQ IDNo. 21) No. 22) HPRT1 RT-PCR TGCTCGAGATG TCCCCTGTTG 192 TGATGAAGGACTGGTCATT (SEQ ID (SEQ ID No. 23) No.24)

Indirect Immunofluorescence. Immunofluorescence assays on cells andtissue sections were performed as previously described (29). Briefly,differentiated samples were fixed using 4% (w/v) paraformaldehyde andmembrane permeabilization (for cells only) was done with 0.3% (w/v)Triton-X 100 (Sigma). Unspecific sites were blocked using 2% BSA for 30min prior to incubation with rabbit polyclonal anti-PDX1 (Millipore,06-1379, 1:1000), rabbit anti-C-Peptide (Cell Signaling, 4593, 1:500,Danvers, Mass.) and mouse monoclonal anti-Glucagon (Abcam, ab10988,1:200) primary antibodies. Phalloidin-TRITC (Millipore, FAK100, 1:1000,Billerica, Mass.) was used to stain the actin stress fiber network(F-actin). Nuclei were stained using DAPI contained in Vectashield®mounting medium for fluorescence (Vector labs, Burlingame, Calif.).Quantitation of C-peptide positive stained cells and islet surface areawas performed using NIS-Elements Advanced Research software (Nikon).

Western blot analysis. Proteins were extracted from mouse pancreas,separated by SDS-PAGE, and transferred onto nitrocellulose membranes aspreviously described (30, 31). Membranes were blocked in PBS containing5% skim milk and 0.1% TWEEN 20 (BioRad, Hercules, Calif.). Rabbitanti-C-Peptide (Cell Signaling, 4593, 1:500), mouse monoclonalanti-Glucagon (Abcam, ab10988, 1:500) and anti-actin (Millipore,MAB1501, 1:2000) primary antibodies were incubated with membranesovernight at 4° C.

Mouse Blood Glucose Assays. C57BL/6 WT and RARβ KO mice were used forthis experiment as previously described (26). Briefly, mice were fastedfor 15 hours overnight and a 50% dextrose solution (2 g/kg body weight)was injected intraperitoneally. Blood glucose levels were measured fromthe tail vein at 0, 15, 30, 60, and 120 min using the One Touch BloodGlucose Monitoring System (LifeScan) (32).

Statistical analysis. All experiments were performed at least 3 times(n>3) using independent biological triplicates. Results were presentedas means±SEM. All statistical tests were performed using GraphPad InStatsoftware version 3.10. A p-value of <0.05 indicated statisticalsignificance.

Example 2—Pancreatic Differentiation and Assessment of PancreaticMarkers in WT Mouse ES Cells

The endocrine differentiation protocol was selected because it includedRA-treatment at day 7 and also showed expression of later stageendocrine markers using human ES cells (15). The D'Amour et al. (2006)pancreatic differentiation protocol was used with some slightmodifications to generate pancreatic endocrine cells in culture throughthe use of specific growth factors (FIG. 1A). The first modificationreplaced Wnt3a with BIO-acetoxime (BIO). Wnt3a has been documented asbeing important for mesendoderm specification and BIO-acetoxime is aselective inhibitor of GSK-3β which indirectly acts as a Wnt3a agonistduring cell differentiation (33, 34). Second, nicotinamide was includedduring the last stage of differentiation because various publishedprotocols included this reagent due to strong evidence for its efficacyin pancreatic differentiation (35, 36).

To characterize the impact of the differentiation protocol on pancreaticendocrine specification in WT ES cells, cellular extracts were harvestedat various time points during the procedure (FIG. 1B). The mRNA levelsof various differentiation markers were assessed by RT-PCR for thedifferent experimental conditions. LIF withdrawal combined with theaddition of BIO and Activin A to the culture system caused a drasticdecrease in the levels of ES cell markers Nanog and Rex1 (FIG. 1B, lane6) compared to untreated and RA-treated ES cells (FIG. 1B, lanes 1 to4). Such a phenomenon was observed throughout the subsequent phases ofthe differentiation protocol (FIG. 1B, lanes 7 to 12). While robustexpression of glucagon, a functional marker of α-cells (37), wasobserved by day-14 (FIG. 1B, lane 8), somatostatin, a hormone secretedby δ-cells (37), was detectable as early as day-5 (FIG. 1B, lane 6).Insulin-1 (β-cell marker)(37) was detected by day-11 of thedifferentiation protocol but its expression fluctuated depending on theuses of HGF, IGF1, or both factors together during the endocrine celldifferentiation stage (FIG. 1B, lanes 9 to 11). The most consistentexpression of all 3 pancreatic endocrine differentiation markers testedwas observed by combining HGF and IGF1 with nicotinamide, from day-14 to17 (FIG. 1B, lane 12). Even though keeping ES cells in culture, atconfluence and in absence of LIF for 17 days, caused a decrease in Nanogand Rex1 expression, such conditions failed to induce any of thedifferentiation markers tested (FIG. 1B, lane 5).

These observations confirm the conversion of ES cells to endocrine cellsable to express pancreatic hormone-encoding genes, according to a methoddescribed previously (15). Such a biological model represents a powerfultool to investigate the role of RARβ at specific stages of pancreaticendocrine differentiation.

Example 3—RARβ Knockout Delays Pdx1 Expression in Pancreatic EndocrineDifferentiation

As previously mentioned, the RA signaling, including the participationof RARβ, was suggested to be crucial for the onset of pancreaticendocrine differentiation (11, 20, 21, 24). In order to study thespecific role of RARβ in such a process, WT and RARβ KO mouse ES cellswere subjected to the endocrine differentiation protocol describedabove. RT-PCR analysis confirmed the absence of RARβ transcript in KOcells (FIG. 2A). The RARβ2 isoform, like Cyp26a1, represents aRA-inducible gene (38). This explains why stronger RARβ signal wasobserved in the presence of RA, in WT cells compared to untreated ones(FIG. 2A). RA-dependent Cyp26a1 expression was observed in both WT andRARβ KO ES cells, suggesting that KO cells are still responding to RAstimuli (FIG. 2A). Using this model of RARβ deletion, the inventorssought to determine the impact of such a retinoid receptor on theexpression of Pdx1, which consists in a master regulator of pancreaticcell fate (39-41).

WT and RARβ KO ES cells were differentiated into pancreatic endocrinecells, as described in FIG. 1, and indirect immunofluorescence stainingfor Pdx1 was performed at the different stages of the protocol (FIG.2B). Pdx1 expression was observed in WT differentiating cells by day-5,and was still present at all the other stages tested in a heterogeneouspattern (FIG. 2B). In contrast, Pdx1 protein was absent from nuclei ofdifferencing cells at day-5 and 11, and was only detected by day-14 ofthe protocol in RARβ null cells (FIG. 2B).

These observations suggest that the absence of RARβ in this cell culturesystem undergoing pancreatic differentiation engenders a delay in theinduction of Pdx1, which could potentially affect subsequent key stepsof endocrine specialization.

Example 4—Absence of RARβ Expression Impairs the Global PancreaticEndocrine Differentiation Process

Considering the finding that RARβ deletion in ES cells delays theexpression of Pdx1 during their specialization into pancreatic endocrinecells, the inventors decided to further investigate the impact of such aphenomenon on early, intermediate, and late molecular genetic eventsthroughout the differentiation process. As reported in many studies onreprogramming, decreased expression of pluripotency factors, includingNanog, in ES cells is essential for proper differentiation (42). Nanoglevels were previously shown to decrease around day-5 during thepancreatic endocrine differentiation protocol (FIG. 1B). A comparison ofNanog transcript levels in WT and RARβ KO differentiating cells, showeda sustained expression of this pluripotency factor in KO cells while itis severely repressed in WT controls (FIG. 3A). On the other hand, theexpression of Neurogenin-3 (Ngn3), a master transcription factor duringonset of pancreatic endocrine lineages (39, 41, 43), displayed a phasedinduction pattern in WT cells but was not induced in RARβ knockout (FIG.3A).

Like Ngn3, Paired-box 6 (Pax6) and Islet1 (Isl-1) represent twoimportant transcription factors in pancreatic islet celldifferentiation, which are expressed from intermediate (““mid””) toterminally differentiated (““late””) stages (39, 40, 44, 45). While nodifference were noted for Pax6 expression patterns, Isl-1 displayed adelayed expression peak in RARβ KO cells as compared to WT (day-14versus day-11) (FIG. 3B).

Finally, the expression of different functional endocrinedifferentiation markers such as, glucagon (Gcg; α-cells), insulin-1(Ins1; β-cells) and islet amyloid polypeptide (IAPP; β-cells) wasanalyzed in RARβ KO and WT differentiating cells (15, 46, 47) (FIG. 3C).In all cases, RARβ KO cells showed impaired expression of thosefunctional markers as compared to WT (FIG. 3C). Specifically, by day-17Gcg, Ins1, and Iapp respectively presented ˜5-fold (p=0.04), ˜120-fold(p=0.013), and ˜7-fold (p=0.0002) increases in WT differentiated cellsas compared to RARβ KO (FIG. 3C). Somatostatin (Sst), a functionalmarker of δ-cells (37) also displayed a decreased expression in RARβdeficient cells (not shown).

Taken together, these observations show that RARβ and retinoid signalingplay a central role in pancreatic endocrine differentiation byregulating the expression of certain master genes at early andintermediate stages of the specialization process, which as a resultimpairs the expression of functional markers of pancreatic islet cells.

Example 5—Deletion of RARβ Affects In Vivo Glucose Metabolism andPancreatic Islet Functionality

The tissue culture system used to study diverse steps of pancreaticendocrine differentiation provided important insights about the roleplayed by RARβ in such a physiological process. Specifically, theabsence of RARβ expression leads to decreased or delayed expression ofcrucial transcription factors involved in islet cell differentiation, aswell as decreased expression of functional differentiation markers(FIGS. 2 and 3). Thus, the inventors sought to validate the relevance ofthis finding in an in vivo model. A classical KO of both RARβ alleles inmice, generated and characterized by Ghyselinck et al. (26), was used tostudy the impact of such a deletion on pancreatic endocrine functions.By extracting pancreas from WT and RARβ-deficient mice, and performingindirect immunofluorescence staining for C-peptide, a by-product ofinsulin biosynthesis (48), and glucagon, the inventors observed adecrease (˜75%, p<0.0001) in the size of KO mice islets as compared toWT (FIG. 4A). Western blot analysis confirmed the decrease in C-peptideand glucagon expression in RARβ KO mice pancreas extracts as compared toWT controls (FIG. 4A). These observations demonstrate that RARβ KO micedisplay decreased pancreatic endocrine islet cell production and/ormaintenance, which could have major, deleterious effects on glucosemetabolism.

To assess the systemic effects of RARβ deletion on reduced insulin andglucagon-producing cells, mice of both groups were fasted for 15 hoursand blood glucose concentration was measured. While blood glucose levelsin WT were normal (between 70 and 105 mg/dL) (49), RARβ KO animals werefound to be in a hypoglycemic state, slightly below normal levels(61±4.7 mg/dL) (FIG. 4B) (50). In order to test the functionality ofβ-cells in both mice groups, a time-course blood glucose readingexperiment was performed which an intraperitonial injection of 2 mg/Kg(body weight) dextrose at time ““0””. Then, blood glucose clearance wasmonitored at 0, 15, 30, 45, 60, and 120 minutes in WT and RARβ KO mice.We observed that blood glucose was metabolized faster in WT mice, ascompared to KO (FIG. 4B). Moreover, the average blood glucose levels inRARβ KO mice 120 min after the dextrose injection was significantlyhigher (˜30%, p=0.014) than in WT group, suggesting a lower glucosetolerance in animals lacking such a retinoid receptor (FIG. 4B).

As described in the Examples, by using an ES cell-based directeddifferentiation system (Examples 2-4) and an in vivo gene knockout model(Example 5), the inventors demonstrated the crucial role for RARβ inproper pancreatic endocrine cell differentiation. In both cases, theabsence of RARβ led to a decrease in terminal differentiation andfunctional markers, such as insulin and glucagon production. In mice,RARβ deletion resulted in impaired glucose metabolism, characterized byhypoglycemia and glucose intolerance. Taken together, these findingsindicate that reduced RARβ and retinoic acid signaling are key factorsin glucose metabolism disorders, such as diabetes mellitus type I andII. Hence, the administration of agonists of the RARβ receptor canprevent or treat such disorders.

The study described in Example 2 leads to the conclusion that Pdx1expression, during the pancreatic differentiation process, was delayedin the absence of RARβ (FIG. 2). Such a transcription factor representsa key player in the early determination of pancreatic progenitors andbud expension (39, 40, 51, 52). A previous study reported that RAdirectly induces Pdx1 expression in ES cells (51). Strengthening such astatement, ChIP-chip analyses performed on F9 teratocarcinoma cellsrevealed the presence of a putative retinoic acid response element(RARE) located at ˜3 kb upstream of the transcription start site of Pdx1(not shown). That Pdx1 expression is delayed but not fully suppressed inRARβ-null ES cells opens a door on possible compensatory mechanismsexerted by other RARs. It has been previously noted that RARβ transcriptlevels are increased at stages of endocrine differentiation, while apeak of RARα expression is associated with late differentiation stages(24). Possibly RARα and β together participate in the Pdx1 biphasicexpression pattern, as reviewed by Soria (39). Thus, suppressing RARβwould result exclusively in a late Pdx1 expression as observed intreated RARβ KO cells (FIG. 2).

Pdx1 mis-expression was previously associated with severe β-celldysfunction and increased cell death (53). Accordingly, RARβ KO caused areduction in β-cell terminal differentiation markers' expression, suchas Ins1 and Iapp in the cell culture system (FIG. 3), as well as adecreased number of C-peptide expressing cells in RARβ null-micepancreatic islets (FIG. 4). Recent findings by Dalgin et al. (54) alsolinked RA signaling and endocrine cell fate. Although the authorsclaimed that β-cell progenitors differentiate as α-cells in RAdownstream target mnx1 morphants, the data reported here suggest thatRARβ KO induces a decrease in α-cell differentiation, characterized byreduced expression of glucagon in the cell culture system (FIG. 3) andRARβ null mice (FIG. 4). Thus, the effect observed on islet cells in theabsence of RARβ could be attributed to the role of RA signaling in earlypancreatic differentiation events rather than lineage-specific terminaldifferentiation.

Like Pdx1, the bHLH transcription factor Neurogenin3 (Ngn3) constitutesanother key player in the commitment of endoderm to pancreaticprecursors (40, 43, 47). Among the cascade of transcription factorsinvolved in pancreas development, Ngn3 is the earliest to be expressedin the endocrine differentiation pathway (40, 55). While no linksbetween RA signaling and Ngn3 expression was reported in the literature,RARβ KO cells displayed decreased levels of this transcription factorduring pancreatic differentiation (FIG. 3). Thus, the impact of RARβdeletion on Ngn3 could be indirect and involving the participation ofintermediate factors.

Pax6 and Isl-1 represent two major transcription factors having a rolein endocrine lineage specification after bud formation (45, 56)Considering that Pax6 expression is not affected by RARβ KO, and thatthe Isl-1 peak of expression is only delayed by such a deletion, itappears that absence of RA signaling through RARβ is insufficient tocompletely abrogate endocrine differentiation, but may lead tosignificant defects in islet cell function.

The observations reported here indicate that the absence of RARβexpression impairs development and maintenance of pancreatic islets invivo (FIG. 4). In mammals, glucose intolerance is characterized bysustained high blood glucose levels (above 140 mg/dL) during at leasttwo hours, while hypoglycemia is decreed when blood concentration goesbelow 70 mg/dL (50, 57). Blood glucose assessment 1) after 15 h fastingand 2) upon dextrose injection led us to suggest that RARβ-null micehave a predisposition to fasting hypoglycemia and increased glucoseintolerance, two conditions associated with diabetes mellitus (58).

Close correlations have been made between dietary habits and diabetes,especially for type II (59). Considering the role of RARβ in pancreaticendocrine cell differentiation, and that the RARβ gene itself isup-regulated by retinoic acid, a sustained vitamin A deficient dietcould lead to insufficient islet cell turnover, and eventually todiabetes. RARβ expression is also known to depend on epigeneticregulation (60, 61). For instance, aberrant hypermethylation of variouspromoter elements was reported in different pancreatic disorders such ascancer, diabetes, and chronic pancreatitis (62-64). Therefore,epigenetic silencing of RARβ or other associated effectors could play arole in the onset of certain cases of diabetes.

The production of insulin secreting endocrine cells from ES cells usingRA-based protocols is proposed as a promising tool for diabetic therapy(9). However, ensuring accurate vitamin A consumption and proper RAsignaling via RARβ represent new avenues to prevent or treat diabeticdisorders. In particular, the administration of an RARβ agonist would bea specifically targeted method of enhancing this RARβ signaling toprevent or treat diabetic disorders. Taken together, these findings shedlight on the role of RARβ in pancreatic endocrine differentiation, whichconsequently affects in vivo blood glucose metabolism.

Example 6—RARβ Agonist Treatment Preparation

Preparation of AC261066 (a RARβ agonist from Tocris) solution. AC261066was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 1.5mg/ml and 3.0 mg/ml, and diluted in the drinking water for mice to thefinal concentration of 1.5 mg/100 ml and 3.0 mg/100 ml.

Mice, diet, and drug treatment. WT male C57/BL6 male mice weremaintained on either a standard laboratory chow-fed diet (CFD) with 13%kcal fat, (diet #5053, Lab Diet, Inc, St. Louis, Mo.) or a high fat,western style diet (HFD) with 60% kcals from fat, (diet #58126, LabDiet, Inc., St. Louis, Mo.) for 4 months. One month after the start ofthe high fat diet treatment, the high fat diet group was further splitinto 2 groups for 3 months: i) high fat diet and the drinking watercontaining 1% DMSO; ii) high fat diet and the drinking water containing1.5 mg/100 ml AC261066, a specific RARβ agonist. Then mice weresacrificed by cervical dislocation. Blood and various tissue sampleswere harvested.

Example 7—Pancreas

Semi-Quantitate PCR. Total RNA was extracted from mouse tissues usingTRIzol reagent (Life technologies) and (1 μg) was used to synthesizecDNA. cDNA synthesis was performed at 42° C. for 1 h in a final volumeof 20 μl using qScript (Quanta, MD). Semi-quantitative PCR wereperformed Taq DNA polymerase (Invitrogen, CA). Three step PCR was run asfollows: 94° C. for 30 s, 58-64° C. for 45 s for primer annealing and72° C. for 1 min for primer extension. The number of cycles for eachprimer pair for amplification in the linear range was determinedexperimentally. PCR products were resolved on 2% agarose gels andvisualized by staining with ehtidium bromide. Primers for geneexpression used were as follows:

RARβ2, F: (SEQ ID No. 25) 5′-TGGCATTGTTTGCACGCTGA-3′, R: (SEQ ID No. 26)5′-CCCCCCTTTGGCAAAGAATAGA-3′, CYP26A1, F: (SEQ ID No. 27)5′-CTTTATAAGGCCGCCCAGGTTAC-3′, R: (SEQ ID No. 28)5′-CCCGATCCGCAATTAAAGATGA-3′, LRAT, F: (SEQ ID No. 29)5′-TCTGGCATCTCTCCTACGCTG-3′, R: (SEQ ID No. 30)5′-GTTCCAAGTCCTTCAGTCTCTTGC-3′, INS2, F: (SEQ ID No. 31)5′-TGTGGGGAGCGTGGCTTCTTCT-3′, R: (SEQ ID No. 32)5′-CAGCTCCAGTTGTGCCACTTGT-3′, HPRT, F: (SEQ ID No. 33)5′-TGCTCGAGTGTGATGAAGG-3′, R: (SEQ ID No. 34) 5′-TCCCTGTTGACTGGTCATT-3′.

Analysis of pancreatic retinoids. The frozen pancreas tissue samples(˜100 mg) were homogenized in 500 μl cold phosphate-buffered saline(PBS). In addition, 100 μl serum was diluted in cold PBS to total volumeof 500 μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millennium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

4-hydroxynonenal (4-HNE) staining. Paraffin-embedded sections (from twoto four mice per group) were deparaffinized and rehydrated, and antigenretrieval was performed using an antigen unmasking solution (VectorLaboratories, H-3300). After quenching endogenous peroxidase with 3%H₂O₂, the tissue sections were blocked with the blocking reagent (fromthe M.O.M. kit from Vector Laboratories). Then, tissue sections wereincubated with a 4-HNE antibody (1:400; mouse monoclonal antibody;Abcam, ab48506) overnight at 4° C. The sections were then incubated withsecondary antibodies (1:200, anti-mouse IgG from the M.O.M kit). As anegative control, sections were stained without incubation with primaryantibodies. The signals were visualized based on a peroxidase detectionmechanism with 3,3-diaminobenzidine (DAB) used as the substrate.

Retinoid levels in pancreatic tissue. Our HPLC analysis revealed thatthat pancreata from HF-fed obese mice had dramatically decreased levelsretinol (VA, vitamin A) compared to CF (control diet) controls (FIG. 5).Retinyl palmitate was undetectable in pancreata tissue from HF-fed mice(FIG. 5), showing profound pancreas vitamin A deficiency.

Serum retinol from mice on a high fat diet vs. control diet compared tothe pancreas retinol and retinyl palmitate levels from mice on a highfat vs. control diet. The serum retinol levels are similar or a bithigher in the HF diet mice, but the pancreas retinol levels are muchlower in the HF diet mice, showing vitamin A deficiency in the pancreaseven in the presence of normal serum vitamin A (FIG. 6).

AC261066 decreases oxidative stress levels in the pancreas from HF-fedmice. High fat diet results in excessive reactive oxygen species (ROS)production that triggers inflammatory responses and subsequent injuriesin many tissues. Therefore, we examined the levels of 4-hydroxynonenal(4-HNE), an α,β-unsaturated hydroxyalkenal that is produced by lipidperoxidation in cells during oxidative stress, and is a marker ofoxidative stress caused by reactive oxygen species (ROS) in thepancreas. The pancreatic islets from HF-fed mice showed an increase inthe 4-HNE levels compared to the chow-fed controls (FIG. 7). Thepancreatic islet samples from the high fat diet plus AC261066 groupexhibited markedly lower 4-HNE staining intensity levels compared toHF-vehicle treated mice (FIG. 7).

AC261066 does diminish pancreatic islet insulin expression. Next weexamined the changes to pancreatic expression of endocrine hormones inCF, HF and HF+AC261066 fed mice. Pancreatic islets stained forpro-insulin c-peptide (green) and glucagon (red) revealed that isletsfrom HF and HF+AC261066 fed mice showed a marked increase in c-peptidestaining compared to control diet controls (FIG. 8). AC261066 slightlydecreased c-peptide level in the HF diet mice.

AC2621066 increased pancreatic mRNA expression of RARβ in obese andvitamin A deficient mice. Consistent with our HPLC data demonstratingthat pancreata tissue from HF-fed, obese mice had significantlydecreased VA (vitamin A) levels, and significantly decreased mRNA levelsof the VA responsive gene and VA signaling transcription factor, RARβ.RARβ was decreased in pancreata of HF-fed obese mice compared to controldiet fed mice (FIG. 9). mRNA levels of RARβ in pancreata HF-AC261066treated mice were increased compared to HF-vehicle treated mice (FIG.9), and near levels observed in non-obese controls, suggesting thatAC261066 can prevent or reverse the loss of VA signaling in VA depletedtissue. Similar findings in vitamin A deficient mice, FIG. 10.

Example 8—Liver

Hematoxylin and Eosin Staining. At sacrifice, fresh mouse liver sampleswere fixed in 4% formaldehyde solution for 24 hr and embedded inparaffin blocks. Liver paraffin sections were cut 5 microns thick andmounted on glass slides and stained with hematoxylin and eosin (H and E)using standard protocols.

Combined oil red O and Immunofluorescence. Staining. Fresh mouse liversamples were embedded in optimal cutting temperature (OCT) medium andimmediately frozen to −70 centigrade. Cryosections were then fixed in 4%formaldehyde for 1 hr at room temp. Slides were then rinsed three timesin deionized water (dH2O) for 30 s, followed by treatment with 0.5%Triton X-100 in PBS for 5 min. Sections were then washed three timeswith PBS for 5 min. Samples were with incubated 2% bovine serum albumin(BSA) for 30 min at room temperature to block for unspecific antibodybinding. Following blocking, sections were washed three times in PBS andincubated with mouse monoclonal antibody against α-SMA (1:500) (Dako,Inc) for 24 h at 4° C. After 24 h sections were washed three times in PBand incubated with Alexa-Flour-488 anti-mouse secondary anti-body(1:500) (Invitrogen, Inc) for 30 min at room temperature. Sections werethen washed three times in PBS and incubated with working strengthoil-red O solution for 30 minutes at room temperature. Sections werethen rinsed for 30 minutes under running tap water and cover-slippedwith Vectashield hard mount plus DAPI (Vector Labs, Inc).

Semi-Quantitate PCR (Liver). Total RNA was extracted from mouse tissuesusing TRIzol reagent (Life technologies) and (1 μg) was used tosynthesize cDNA. cDNA synthesis was performed at 42° C. for 1 h in afinal volume of 20 μl using qScript (Quanta, MD). Semi-quantitative PCRwere performed Taq DNA polymerase (Invitrogen, CA). Three step PCR wasrun as follows: 94° C. for 30 s, 58-64° C. for 45 s for primer annealingand 72° C. for 1 min for primer extension. The number of cycles for eachprimer pair for amplification in the linear range was determinedexperimentally. PCR products were resolved on 2% agarose gels andvisualized by staining with ehtidium bromide. Primers for geneexpression used were as follows:

RARβ2, F: (SEQ ID No. 25) 5′-TGGCATTGTTTGCACGCTGA-3′, R: (SEQ ID No. 26)5′-CCCCCCTTTGGCAAAGAATAGA-3′, CYP26A1, F: (SEQ ID No. 27)5′-CTTTATAAGGCCGCCCAGGTTAC-3′, R: (SEQ ID No. 28)5′-CCCGATCCGCAATTAAAGATGA-3′, LRAT, F: (SEQ ID No. 29)5′-TCTGGCATCTCTCCTACGCTG-3′, R: (SEQ ID No. 30)5′-GTTCCAAGTCCTTCAGTCTCTTGC-3′, INS2, F: (SEQ ID No. 31)5′-TGTGGGGAGCGTGGCTTCTTCT-3′, R: (SEQ ID No. 32)5′-CAGCTCCAGTTGTGCCACTTGT-3′, TNFα, F: (SEQ ID No. 35)5′-CCTGTAGCCCACGTCGTAG-3′, R: (SEQ ID No. 36)5′-GGGAGTAGACAAGGTACAACCC-3′, MCP1, F: (SEQ ID No. 37)5′-TTAAAAACCTGGATCGGAACCAA-3′, R: (SEQ ID No. 38)5′-GCATTAGCTTCAGATTTACGGGT-3′, HPRT, F: (SEQ ID No. 33)5′-TGCTCGAGTGTGATGAAGG-3′, R: (SEQ ID No. 34) 5′-TCCCTGTTGACTGGTCATT-3′.

Serum triglyceride level measurement. The analysis of serum triglyceridelevels was carried out using a bichromatic assay at the Laboratory ofComparative Pathology of the Memorial Sloan-Kettering Cancer Center.Chow-fed diet (CFD) n=2; high fat diet (HFD) n=3; high fat diet+AC261066(HFDAC) n=5.

Analysis of serum and liver retinoids. The frozen liver tissue samples(˜100 mg) were homogenized in 500 μl cold phosphate-buffered saline(PBS). In addition, 100 μl serum was diluted in cold PBS to total volumeof 500 μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millennium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

4-hydroxynonenal (4-HNE) staining. Paraffin-embedded sections (from twoto four mice per group) were deparaffinized and rehydrated, and antigenretrieval was performed using an antigen unmasking solution (VectorLaboratories, H-3300). After quenching endogenous peroxidase with 3%H2O2, the tissue sections were blocked with the blocking reagent (fromthe M.O.M. kit from Vector Laboratories). Then, tissue sections wereincubated with a 4-HNE antibody (1:400; mouse monoclonal antibody;Abcam, ab48506) overnight at 4° C. The sections were then incubated withsecondary antibodies (1:200, anti-mouse IgG from the M.O.M kit). As anegative control, sections were stained without incubation with primaryantibodies. The signals were visualized based on a peroxidase detectionmechanism with 3,3-diaminobenzidine (DAB) used as the substrate.

Analysis of serum and liver retinoids. The frozen liver tissue samples(˜100 mg) were homogenized in 500 μl cold phosphate-buffered saline(PBS). In addition, 100 μl serum was diluted in cold PBS to total volumeof 500 μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millenium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

AC261066 diminished hepatic steatosis. H and E staining of liversections from treatment mice revealed that 4 months of a HF westernstyle diet lead to increased hepatocyte lipid accumulation in HF-fedmice compared to CFD-fed mice (FIG. 11). HF-fed mice treated withAC261066 showed marked decreased hepatocyte lipid infiltration comparedto HF-vehicle treated mice (FIG. 11). HF-fed mice treated with a RAR γligand (CD1530) showed no decrease in hepatic lipid accumulation (FIG.11).

AC261066 diminishes hepatic gene expression of alpha-SMA (alpha-smoothmuscle actin) and SREBP1c. Consistent with our immunofluorescencemicroscopy showing that α-SMA protein is decreased in HF-AC261011 fedmice compared to HF-vehicle controls, hepatic mRNA levels of alpha-SMAwere also decreased in livers of HF-AC261011 fed mice, but not in thelivers of HF-CD1530 treated mice (FIG. 12). We also measured mRNAexpression of SREBP1-c, which codes for a transcription factorresponsible for de novo synthesis of triglyceride and is oftenover-expressed in livers of animals with experimentally induced NAFLD.Our analysis revealed that mRNA levels of SREBP1-c are markedly higherin livers of HF-fed and HF-fed CD1530 treated mice, but not in livers ofHF-AC261011 treated mice (FIG. 12).

AC261066 diminishes hepatic stellate cell (HSC) activation. Liversections co-stained with the neutral lipid stain oil-red-o were inagreement with the H and E staining, demonstrating that HF-fed obesemice had ectopic accumulation of hepatic lipids (red) compared to CFcontrols (FIG. 13). Livers of HF-AC261066-fed mice had marked diminishedhepatic lipid accumulation compared to HF vehicle-fed mice (FIG. 13).This effect was not observed in the livers of HF-fed mice treated withthe CD1530 (RARγ agonist).

Activated HSCs contribute to normal liver tissue repair processes, butunresolved HSC activation can lead to fibrotic lesion formation and theprogression of steatosis to advanced NAFLD, such as non-alcoholicsteatohepatitis (NASH). To examine whether HF-fed obese mice exhibitedevidence of increased activation of HSCs we stained liver sections withan α-SMA antibody. This analysis revealed the livers of HF-fed mice hadincreased α-SMA positive (green) staining compared to lean, CF controls.α-SMA positive areas tended to cluster in areas with hepatocyte lipidinfiltration (FIG. 13). Compared to HF-fed mice, livers ofHF-fed-AC261066 treated mice had decreased intensity and regions ofα-SMA positive staining (FIG. 13). Moreover, clustering a-SMA in lipidpositive (red) regions was not observed in liver of HF-AC261066 treatedmice. Livers of HF-fed CD1530 treated mice had no evidence decreasedlipid accumulation or α-SMA expression intensity or patterns compared toHF fed-vehicle treated mice.

AC261066 diminishes hepatic gene expression of pro-inflammatorymediators. NAFLD is typically associated with increased hepaticexpression of pro-inflammatory cytokines and mediators such as themonocyte chemokine MCP-1 and the cytokine TNF-α. We examined expressionof these genes in livers of CF and HF-fed mice. Our analysis revealedthat mRNA levels of both MCP-1 and TNF-α were markedly elevated inlivers of HF-fed mice HF-fed CD1530 treated mice, but not in livers ofHF-fed AC261066 treated mice (FIG. 14).

AC261066 does not elevate serum triglyceride levels. We examined thetriglyceride levels in mouse serum samples because elevatedtriglycerides are a risk factor for cardiovascular disease. As shown inFIG. 15, HF or HF+AC261066 feeding does not affect serum triglyceridelevels compared CF controls. This suggests that AC261066 does notincrease risk for cardiovascular disease and suggests that the liverlipid lowering effect of AC261066 does not correlate with increasedhepatic lipid export.

AC261066 partially reverses depletion of VA in livers of HF-fed ObeseMice. The liver stores approximately 80-90% of total body VA, thereforewe conducted HPLC to determine the tissue levels of the major storageform of VA, retinyl-palmitate and of all-trans retinol in lean CF, HFand HF+AC261066 fed mice. Our analysis revealed that levels ofretinyl-palmitate and retinol were decreased by 97% and 92% in livers inHF-fed, obese mice compared to lean, CF controls (FIG. 16). Serum levelsof the major circulating form of VA, all-trans retinol were notdifferent between CF, HF and HF+AC261066 fed mice, suggesting thatHF-driven obesity leads to tissue VA depletion which is not reflected byserum VA levels.

Livers of mice fed HF+AC261066 and CD1530 also had significantly loweredretinyl palmitate and retinol compared to controls, however compared toHF-vehicle treated mice, we observed 55% higher levels of retinylpalmitate in the livers from HF-AC261066 fed mice, while retinylpalmitate levels in the liver of HF+CD1530 treated mice were almost 48%lower than livers from HF-vehicle treated mice (FIG. 16). This suggeststhat longer administration of AC261066 to HF-fed obese mice may havesignificantly reversed HF-obesity driven liver VA depletion.

Oxidative stress level, as assessed by 4-hydroxynoneal (4-HNE), is lowerin the liver from the high fat diet plus AC261066 group than that in thehigh fat diet group. High fat diet results in excessive reactive oxygenspecies (ROS) production that triggers inflammatory responses andsubsequent injuries in many tissues. Therefore, we examined the levelsof 4- hydroxynonenal (4-HNE), an α,β-unsaturated hydroxyalkenal that isproduced by lipid peroxidation in cells during oxidative stress, and isa marker of oxidative stress caused by reactive oxygen species (ROS) inthe liver. The liver from the high fat diet group showed a largeincrease in the 4-HNE levels compared to the control fat diet group, andthe liver samples from the high fat diet plus AC261066 group exhibitedlower 4-HNE levels than those from the high fat diet group (FIG. 17).

Example 9—Kidney

Hematoxylin and Eosin Staining. At sacrifice, fresh mouse liver sampleswere fixed in 4% formaldehyde solution for 24 hr and embedded inparaffin blocks. Kidney paraffin sections were cut 5 microns thick andmounted on glass slides and stained with hematoxylin and eosin (H and E)using standard protocols.

Combined oil red O and Immunofluorescence staining. Fresh mouse kidneysamples were embedded in optimal cutting temperature (OCT) medium andimmediately frozen to −70 centigrade. Cryosections were then fixed in 4%formaldehyde for 1 hr at room temp. Slides were then rinsed three timesin deionized water (dH2O) for 30 s, followed by treatment with 0.5%Triton X-100 in PBS for 5 min. Sections were then washed three timeswith PBS for 5 min. Samples were with incubated 2% bovine serum albumin(BSA) for 30 min at room temperature to block for unspecific antibodybinding. Following blocking, sections were washed three times in PBS andincubated with mouse monoclonal antibody against α-SMA (1:500) (Dako,Inc) for 24 h at 4° C. After 24 h sections were washed three times in PBand incubated with Alexa-Flour-488 anti-mouse secondary anti-body(1:500) (Invitrogen, Inc) for 30 min at room temperature. Sections werethen washed three times in PBS and incubated with working strengthoil-red O solution for 30 minutes at room temperature. Sections werethen rinsed for 30 minutes under running tap water and cover- slippedwith Vectashield hard mount plus DAPI (Vector Labs, Inc).

Semi-Quantitative PCR. Total RNA was extracted from mouse tissues usingTRIzol reagent (Life technologies) and (1 μg) was used to synthesizecDNA. cDNA synthesis was performed at 42° C. for 1 h in a final volumeof 20 μl using qScript (Quanta, MD). Semi-quantitative PCR wereperformed Taq DNA polymerase (Invitrogen, CA). Three step PCR was run asfollows: 94° C. for 30 s, 58-64° C. for 45 s for primer annealing and72° C. for 1 min for primer extension. The number of cycles for eachprimer pair for amplification in the linear range was determinedexperimentally. PCR products were resolved on 2% agarose gels andvisualized by staining with ehtidium bromide. Primers for geneexpression used were as follows:

RARβ2, F: (SEQ ID No. 25) 5′-TGGCATTGTTTGCACGCTGA-3′, R: (SEQ ID No. 26)5′-CCCCCCTTTGGCAAAGAATAGA-3′, CYP26A1, F: (SEQ ID No. 27)5′-CTTTATAAGGCCGCCCAGGTTAC-3′, R: (SEQ ID No. 28)5′-CCCGATCCGCAATTAAAGATGA-3′, TNFα, F: (SEQ ID No. 35)5′-CCTGTAGCCCACGTCGTAG-3′, R: (SEQ ID No. 36)5′- GGGAGTAGACAAGGTACAACCC-3′, HPRT, F: (SEQ ID No. 33)5′-TGCTCGAGTGTGATGAAGG-3′, R: (SEQ ID No. 34) 5′-TCCCTGTTGACTGGTCATT-3′.

Analysis of kidney retinoids. The frozen kidney tissue samples (˜100 mg)were homogenized in 500 μl cold phosphate-buffered saline (PBS). Inaddition, 100 μl serum was diluted in cold PBS to total volume of 500μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millennium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

4-hydroxynonenal (4-HNE) staining. Paraffin-embedded sections (from twoto four mice per group) were deparaffinized and rehydrated, and antigenretrieval was performed using an antigen unmasking solution (VectorLaboratories, H-3300). After quenching endogenous peroxidase with 3%H₂O₂, the tissue sections were blocked with the blocking reagent (fromthe M.O.M. kit from Vector Laboratories). Then, tissue sections wereincubated with a 4-HNE antibody (1:400; mouse monoclonal antibody;Abcam, ab48506) overnight at 4° C. The sections were then incubated withsecondary antibodies (1:200, anti-mouse IgG from the M.O.M kit). As anegative control, sections were stained without incubation with primaryantibodies. The signals were visualized based on a peroxidase detectionmechanism with 3,3-diaminobenzidine (DAB) used as the substrate.

AC261066 diminished renal lipid accumulation. H and E staining of kidneysections from treatment mice revealed that 4 months of a HF westernstyle diet lead to increased renal lipid accumulation in HF-fed micecompared to CFD-fed mice (FIG. 18). HF-fed mice treated with AC261066showed markedly decreased renal lipid infiltration compared toHF-vehicle treated mice (FIG. 18). HF-fed mice treated with a RAR γligand (CD1530) showed no decrease in renal lipid accumulation (FIG.18).

AC261066 diminishes renal expression of alpha-SMA. Kidney sectionsco-stained with the neutral lipid stain oil-red-o were in agreement withthe H and E staining, demonstrating that HF-fed obese mice had ectopicaccumulation of renal lipids (red) compared to CF controls (FIG. 18).Kidneys of HF-AC261066-fed mice had marked diminished hepatic lipidaccumulation compared to HF vehicle-fed mice (FIG. 19). Alpha-SMA isrequired for normal kidney tissue repair processes, but uncheckedalpha-SMA secretion can lead to fibrotic lesion formation and theprogression of advanced renal disease. As expected kidney sectionsstained with the neutral lipid stain oil-red-o (red) showed markedincrease in renal lipid droplets in kidneys of HF-fed mice compared tocontrol fed mice. In agreement with our H and E histological analysis,kidney sections from HF+AC261066 treated mice had comparably less oilred o positive areas. α-SMA (green) staining also revealed that kidneysof HF-fed mice had increased α-SMA positive areas compared to controlfed mice (FIG. 19). This increase in α-SMA positive areas was notobserved in kidneys of HF+AC261066 treated mice.

Retinoid levels in kidneys. Our HPLC analysis of kidney tissuedemonstrated that HF-fed obese mice had significantly decreased levelsof kidney retinyl palmitate and retinol compared to chow fed controls(FIG. 20).

AC261066 diminishes kidney gene expression of pro-inflammatorymediators. Fibrosis is associated increased renal expression ofpro-inflammatory cytokines and mediators. We examined whether kidneys ofHF-fed mice had evidence of inflammation marked by increased expressionof inflammatory cytokines such as TNF-α. Our analysis revealed that mRNAlevels of TNF-α were markedly elevated in livers of HF-fed mice, but notin livers of HF-fed AC261066 treated mice (FIG. 21).

AC261066 increased kidney gene expression of RARβ2. Consistent with theHPLC data demonstrating that VA levels are diminished in kidney ofHF-fed mice, our kidney gene expression analysis revealed that RARβ2mRNA is markedly decreased in the kidney of HF-fed mice (FIG. 21).Kidney's from HF-AC261066 did not have decreased RARβ2 mRNA levels (FIG.21).

Oxidative stress level, as assessed by 4-hydroxynoneal (4-HNE), is lowerin the kidneys from the high fat diet plus AC261066 group than that inthe high fat diet group. High fat diet results in excessive reactiveoxygen species (ROS) production that triggers inflammatory responses andsubsequent injuries in many tissues. Therefore, we examined the levelsof 4-hydroxynonenal (4-HNE), an α,β-unsaturated hydroxyalkenal that isproduced by lipid peroxidation in cells during oxidative stress, and isa marker of oxidative stress caused by reactive oxygen species (ROS) inthe kidneys The kidneys from the high fat diet group showed a largeincrease in the 4-HNE levels compared to the control fat diet group, andthe kidneys from the high fat diet plus AC261066 group exhibited lower4-HNE levels than those from the high fat diet group (FIG. 22).

Example 10—TESTES

Semi-Quantitative PCR. Total RNA was extracted from mouse tissues usingTRIzol reagent (Life technologies) and (1 μg) was used to synthesizecDNA. cDNA synthesis was performed at 42° C. for 1 h in a final volumeof 20 μl using qScript (Quanta, MD). Semi-quantitative PCR wereperformed Taq DNA polymerase (Invitrogen, CA). Three step PCR was run asfollows: 94° C. for 30 s, 58-64° C. for 45 s for primer annealing and72° C. for 1 min for primer extension. The number of cycles for eachprimer pair for amplification in the linear range was determinedexperimentally. PCR products were resolved on 2% agarose gels andvisualized by staining with ehtidium bromide. Primers for geneexpression used were as follows:

RARβ2, F: (SEQ ID No. 25) 5′-TGGCATTGTTTGCACGCTGA-3′, R: (SEQ ID No. 26)5′-CCCCCCTTTGGCAAAGAATAGA-3′, CYP26A1, F: (SEQ ID No. 27)5′-CTTTATAAGGCCGCCCAGGTTAC-3′, R: (SEQ ID No. 28)5′- CCCGATCCGCAATTAAAGATGA-3′, HPRT, F: (SEQ ID No. 33)5′-TGCTCGAGTGTGATGAAGG-3′, R: (SEQ ID No. 34) 5′-TCCCTGTTGACTGGTCATT-3′.

Analysis of testes retinoids. The frozen kidney tissue samples (˜100 mg)were homogenized in 500 μl cold phosphate-buffered saline (PBS). Inaddition, 100 μl serum was diluted in cold PBS to total volume of 500μl. Retinyl acetate was added to each sample before the retinoidextraction for the calculation of extraction efficiency. The retinoidswere extracted into 350 μl of organic solution (acetonitrile/butanol,50:50, v/v) in the dark. The high performance liquid chromatography(HPLC) was performed using a Waters Millennium system (Waters). Eachsample (100 μl of the 350 μl) was loaded onto an analytical 5-μm reversephase C18 column (Vydac, Hesperia, Calif.) and eluted at a flow rate of1.5 ml/min. Two mobile phase gradient systems were used. Retinoids wereidentified by HPLC based on two criteria: an exact match of theretention times of unknown peaks with those of authentic retinoidstandards and identical UV light spectra (220-400 nm) of unknownsagainst spectra from authentic retinoid standards during HPLC by the useof a photodiode array detector. The amounts of retinoids were calculatedfrom the areas under the peaks detected at the wave-length of 325 nm.The levels of retinol and retinyl esters were normalized to the tissueweight.

Retinoid levels in testes. Our HPLC analysis of testes demonstrated thatHF-fed obese mice had significantly decreased levels of retinylpalmitate (storage form of VA) and decreased retinol compared to chowfed controls (FIG. 23).

Testes of HF-fed Mice have decreased expression of VA relevant genesexpression. Consistent with the HPLC data demonstrating that VA levelsare diminished in kidney of HF-fed mice, our testes gene expressionanalysis revealed that RARβ2 and CYP26A1, and RAR gamma2 mRNAs aremarkedly decreased in the testes of HF-fed mice (FIG. 24).

RARβ agonist AC55649 is prepared at a concentration of 3.0 mg/100 ml in1% DMSO and is used to treat mice as described in Examples 6-10.

Example 11—Methods. Preparation of AC261066 (a RARβ Agonist from Tocris)Solution

AC261066(Lund et al., Discovery of a potent, orally available, andisoform-selective retinoic acid beta2 receptor agonist. J Med Chem.2005; 48(24):7517-9) and CD1530 (Thacher et al. Therapeutic applicationsfor ligands of retinoid receptors. Curr Pharm Des. 2000; 6(1):25-58) wasdissolved in dimethyl sulfoxide (DMSO) at the concentration of 1.5 mg/mlfor AC261066 and 2.5 mg/ml for CD1530, and diluted in the drinking waterfor mice to the final concentration of 1.5 mg/100 ml and 2.5 mg/100 ml,respectively.

Example 12—Mice, Diet, and Drug Treatment

WT male C57/BL6 male mice were maintained on either a standardlaboratory chow-fed diet (Con) with 13% kcal fat, (diet #5053, Lab Diet,Inc, St. Louis, Mo.) or a high fat, western style diet (HFD) with 45%kcals from fat, (diet #58126, Lab Diet, Inc., St. Louis, Mo.) for 4months. One month after the start of the HFD treatment, the HFD groupwas further split into 3 groups for 3 months: i) HFD and the drinkingwater containing 1% DMSO; ii) high fat diet (HFD) and drinking watercontaining 1.5 mg/100 ml AC261066, a specific RARβ agonist in 1% DMSO oriii) HFD and drinking water containing 1.5 mg/100 ml CD1530, a specificRARγ agonist in 1% DMSO. Then mice were sacrificed by cervicaldislocation. Blood and various tissue samples were harvested.

Example 13—Serum Triglyceride and Cholesterol Level Measurements

The analysis of serum triglyceride levels was carried out using abichromatic assay at the Laboratory of Comparative Pathology of theMemorial Sloan-Kettering Cancer Center. Chow-fed diet (Con) n=4; highfat diet (HFD) n=4; high fat diet (HFD)+AC261066 n=4; and high fat diet(HFD)+CD1530, n=4.

Example 14—Quantitative Real Time PCR (Q-RT-PCR)

Total RNA was extracted from mouse tissues using TRIzol reagent (Lifetechnologies) and (1 μg) was used to synthesize cDNA. cDNA synthesis wasperformed at 42° C. for 1 h in a final volume of 20 μl using qScript(Quanta, MD). Q-RTPCR was performed as previously described (15).Primers for gene expression used were as follows:

SREBP1C, F: (SEQ ID No. 39) 5′-CAAGGCCATCGACTACATCCG-3′, R:(SEQ ID No. 40) 5′- CACCACTTCGGGTTTCATG-3′, FAS, F: (SEQ ID No. 41)5′-GGAGGTGGTGATAGCCGGTAT-3′, R: (SEQ ID No. 42)5′-TGGGTAATCCATAGAGCCCAG-3′, DGAT1, F: (SEQ ID No. 43)5′-ATGATGGCTCAGGTCCCACT-3′, R: (SEQ ID No. 44)5′-CACTGGGGCATCGTAGTTGA-3′, SIRT1, F: (SEQ ID No. 45)5′-TCTCCTGTGGGATTCCTGAC-3′, R: (SEQ ID No. 46)5′-CTCCACGAACAGCTTCACAA-3′, PPARα, F: (SEQ ID No. 47)5′-AGAGCCCCATCTGTCCTCTC-3′, R: (SEQ ID No. 48)5′-ACTGGTAGTCTGCAAAACCAAA-3′, PPARγ, F: (SEQ ID No. 49)5′-CTCCAAGAATACCAAAGTGCGA-3′, R: (SEQ ID No. 50)5′-GCCTGATGCTTTATCCCCACA-3′, SREBP2, F: (SEQ ID No. 51)5′-GCAGCAACGGGACCATTCT-3′, R- (SEQ ID No. 52)5′-CCCCATGACTAAGTCCTTCAACT-3′, HMGCR, F: (SEQ ID No. 53)5′- AGCTTGCCCGAATTGTATGTG-3′, R- (SEQ ID No. 54)5′-TCTGTTGTGAACCATGTGACTTC-3′, PEPCK. F: (SEQ ID No. 55)5′-TGCCCAAGGCAACTTAAGGG-3′, R- (SEQ ID No. 56)5′-CAGTAAACACCCCCATCGCT-3′, FGF21, F: (SEQ ID No. 57)5′-GTGTCAAAGCCTCTAGGTTTCTT-3′, R- (SEQ ID No. 58)5′-GGTACACATTGTAACCGTCCTC-3′, HPRT, F: (SEQ ID No. 59)5′-TGCTCGAGTGTGATGAAGG-3′, R: (SEQ ID No. 60) 5′-TCCCTGTTGACTGGTCATT-3′.

Example 15—Selective RARβ (e.g., RARβ2) Agonists Effectively Control andReduce Serum Triglyceride and Cholesterol Levels

The highly selective RARβ2 agonist, AC261066, provided at a dose of 1.5mg/100 ml in the drinking water, can significantly lower serumcholesterol and triglycerides in mice fed a high fat diet (HFD) for 4months (FIG. 25A). In contrast, HFD-fed mice treated with a RARγagonist, CD1530, showed no decrease in serum cholesterol andtriglycerides levels (FIG. 25A). Using quantitative real time PCR(Q-RT-PCR) we also measured hepatic mRNA levels of genes involved in denovo fatty acid, cholesterol and glucose synthesis. We demonstrated thatAC261066 significantly reduced hepatic mRNA levels of the lipogenicproteins srebp1-c, fas, dgat1, fgf21, and ppar-γ in HFD-fed mice (FIG.25B). We also demonstrated that AC261066 treatment prevented a largedecrease in the hepatic mRNA levels of sirtl and ppar-α, two proteinsinvolved in beta-oxidation of fatty acids (FIG. 1B). Our gene expressionanalysis also revealed that AC261066 treatments significantly reducedhepatic mRNA levels of srebp-2 and hmgcr (FIG. 25B). Srebp-2 is atranscription factor that increases mRNA levels of hmgcr(3-hydroxy-3-methylglutaryl-coenzyme A reductase, or HMG-CoA reductase),the rate-limiting enzyme in de novo cholesterol synthesis. This effectwas not observed in the livers of HFD-fed mice treated with CD1530 (RARγagonist), showing specificity of AC261066 for RAR β2 (FIG. 1B). Geneexpression of pepck, which is the rate-limiting enzyme ingluconeogenesis, was not affected by either AC261066 or CD1530 (FIG.25B). Together, these data demonstrate that specific agonist activationof the transcription factor RARβ2 results in a significant reduction ofboth serum triglyceride and total cholesterol levels in the high fatdiet fed mice, which coincides with hepatic transcriptional changessupporting suppression of de novo triglyceride and cholesterolsynthesis. Collectively, these data strongly indicate that syntheticRARβ2 agonists are novel cholesterol lowering drugs.

TABLE 4 Gene ID and Abbreviations Mouse Human Gene counterpart ID genegene ID DGAT1, diglyceride acyltransferase 1 13350 8694 FAS, fatty acidsynthase 14104 2194 FGF21, fibroblast growth factor 21 56636 26291HMGCR, 3-hydroxy-3-methylglutaryl- 15357 3156 Coenzyme A reductase HPRT,hypoxanthine guanine 15452 3251 phosphoribosyl transferas PEPCK,phosphoenolpyruvate 18534 5105 carboxykinase 1, cytosolic PPARα,peroxisome proliferator 19013 5465 activated receptor alpha PPARγ,peroxisome proliferator 19016 5468 activated receptor gamma SIRT1,sirtuin 1 93759 23411 SREBP1C, sterol regulatory element 20787 6720binding transcription factor 1c SREBP2, sterol regulatory element 207886721 binding transcription factor 2

Example 16—Retinoic Acid Receptor β (RARβ) Agonists Diminish BodyWeight, Diet Induced Glucose Intolerance and Insulin Resistance in HighFat and Genetic Models of Diabetes

Our metabolic studies showed that both RARβ agonists AC261066 andAC55649 lead to significant reductions in body weights of obese, highfat (HF)-fed mice (FIG. 27A) and in genetically obese and diabetic Ob/Oband Db/Db mice (FIG. 27E). Our metabolic studies also revealed thatadministration of both RARβ agonists lead to a reversal of hyperglycemiain diabetic HF-fed (FIG. 27C, D) and genetically obese diabetic mice(FIG. 27F, G, H). Given that mice treated with RARβ agonists had loweredglucose levels we then sought to determine if RARβ agonists couldimprove insulin secretion and whole body insulin metabolism, both ofwhich are typically altered in individuals with type 2 diabetes (T2D).Our insulin metabolism studies showed administration of RARβ agonists togenetically obese diabetic Ob/Ob and Db/Db mice led to improved insulinmetabolism (FIG. 27L, M). We also found that Ob/Ob mice treated with theRARβ AC261066 had decreased pancreatic insulin secretion, which is aindicator of improved pancreatic function.

Example 17—Retinoic Acid Receptor β (RARβ) Agonists Diminishes theNumber of Large Pancreatic Islets and Pancreatic Insulin Content inOb/Ob and Db/Db Models of Obesity and Diabetes

Early stages of T2D begins with altered insulin metabolism referred toas insulin resistance (IR), which leads to enlargement of the insulinproducing cells of pancreas known as β-cells and overproduction ofinsulin to compensate for the IR. Our metabolic studies revealed thatRARβ agonists could improve IR and as a result decrease pancreaticinsulin secretion. Therefore we then sought to determine if RARβagonists reduce the enlargement of β-cells in HF-fed and geneticallydiabetic mice. Using immunohistochemistry and an anti-body for insulin,we examined pancreas sections and found that RARβ agonists led to thereduction pancreatic β-cells (also known as Islets) and pancreaticinsulin content (FIG. 28A, B). Collectively our metabolic and pancreasstudies demonstrate that RARβ agonists i) reduce obesity in HF-fed angenetically obese Ob/Ob and Db/Db mice, ii) improve glucose intoleranceand peripheral insulin resistance in a dietary and genetic model ofobesity and diabetes.

Example 18—Retinoic Acid Receptor β (RARβ) Agonists Reduce theAccumulation of Liver, Pancreas, Kidney, Muscle and AdiposeTriglycerides in Dietary and Genetic Models of Diabetes

Obesity leads to the accumulation of lipids in organs such as the liver,kidney and adipose tissue. Excessive lipids in organs can alter theirfunction and lead to IR and promote the pathogenesis of T2D. Given thereductions in body weight and improved metabolic profile in mice treatedwith RARβ agonists, we examined the organs of HF and genetically obesediabetic mice for the presence of lipids. Our analysis of Hematoxylinand Eosin stained liver, pancreas, kidney and adipose tissue showed thatHF-fed and genetically obese mice treated with RARβ agonists hadsignificant reductions in lipids (triglycerides) in their liver (FIG.29A[a-e] B, C), pancreas (FIG. 29A[f-j], D), kidneys (FIG. 29A[k-o], E),muscle, (FIG. 29F) and adipose tissue (FIG. 29A[p-t], G).

Example 19—Retinoic Acid Receptor β (RARβ) Agonist AC261066 AltersTissue Expression of Genes Involved in Lipogenesis and MitochondrialOxidation of Lipids

Tissue lipids can be altered by increasing lipid breakdown (catabolism)and/or by diminishing lipid synthesis. With the exception of the liver,most organs do not synthesize lipids and will utilize lipids deliveredthrough the blood. Type 2 diabetes (T2D) is associated with a decreasedability of organs such as muscle to catabolize lipids thus leading tothe accumulation of excess lipids. We examined the expression of genesinvolved in the process of catabolism of lipids known as β-oxidation andin lipid synthesis in liver, pancreas, and adipose tissue. Our geneexpression analysis revealed that liver, pancreas and adipose tissuefrom HFD-fed and genetically obese mice treated with RARβ agonist hadsignificant increases in genes that stimulate β-oxidation of lipidsincluding the transcription factor PPARα, the transporter proteinsCPT1-α and CPT2 and the lipid catabolic enzyme acetyl-CoAacetyltransferase 1 (ACAT1) (FIGS. 30A, C and D). In liver and pancreaswe also detected a significant decrease in expression of genes involvedin the suppression of β-oxidation and stimulation of lipogensis,including the rate limiting enzyme in de novo fatty acid synthesis fattyacid synthase (FASN), the lipogenic transcription factor SREBP1 andacetyl-CoA acetyltransferase 1 (ACC1) (FIGS. 30B, and C). Consistentwith the decreases in body and adipose tissue weights, RARβ agonisttreatment also led to a significant increase in genes responsible foradipocyte lipolysis such as hormone sensitive lipase (HSL) and perilipin(PLIN) (FIG. 30D). Adipose tissue expression of the gene adiponectin(ADIPOQ), which is decreased in individuals with T2D and shown tostimulate oxidation of lipids in peripheral tissue, was significantlyincreased in RARβ-treated HFD-fed and genetically diabetic mice (FIG.30D). Collectively our gene expression studies strongly suggest thatRARβ agonist can regulate pathways in liver, pancreas and adipose tissuethat lead to an increase the β-oxidation of lipids and a decrease inlipogenesis. The ability of AC261066 to increase lipid energy metabolismin organs that are central to glucose-energy metabolism and thepathogenesis of T2D suggests that glucose lowering and insulinsensitizing effects of this RARβ agonist are likely associated withmodulation of lipid energy metabolism.

Example 20—Acute Administration of Retinoic Acid Receptor β (RARβ)Agonist Reverses High Fat Induced Glucose Intolerance and InsulinResistance

We tested if acute administration (8 days) of the RARβ agonist AC261066could ameliorate hyperglycemia and insulin resistance in diabeticHFD-fed mice. Acute administration of AC261066 to HF-fed mice had noeffect on body weights, food or water intake (FIGS. 31A, B and C). Ouracute studies revealed that after 24 hours of administration ofAC261066, HFD-fed mice had significant reductions in random glucoselevels (FIG. 31D) and after 8 days of administration of the AC261066HFD-fed mice had significant reductions in hyperglycemia and insulinresistance (FIGS. 31E and F). Our acute metabolic studies of AC261066demonstrate that this RARβ agonist can acutely improve hyperglycemia andinsulin as effective as those observed with long-term administration ofRARβ agonist. The expression profile of one or more such genes (e.g., aslisted in Table 5 below) may be a therapeutic effect indicator which maybe used to direct therapeutic regimen and doses according to the presentinvention.

Example 21—Methods for Certain Examples

Mice, diet, and drug treatment. Dietary Obesity Studies: Wild type (wt)male C57/BL6 male mice were maintained on either a standard laboratorychow-fed diet (Con) with 13% kcal fat, (diet #5053, Lab Diet, Inc, St.Louis, Mo.) or a high fat, western style diet (HFD) with 45% kcals fromfat, (diet #58126, Lab Diet, Inc., St. Louis, Mo.) for 4 months. Onemonth after the start of the HFD treatment, the Con and HFD groupswerefurther split into 3 additional groups for 4 months to: i) remain oneither Con or HFD and the drinking water containing 1% DMSO; ii) Con orHFD and drinking water containing 3.0 mg/100 ml AC261066, a specificRARβ agonist in 1% DMSO, or iii) Con or HFD and drinking watercontaining 3.0 mg/100 ml of AC55649, another specific RARβ agonist in 1%DMSO. All mice remained on their diets for 4 months. After 4 months micewere subjected to metabolic studies and then sacrificed by cervicaldislocation and tissues were snap frozen at −70°C. for future RNAisolation and histology. Genetically obese mice studies: Lep^(−0b) andLepr-^(db) mice commonly referred to as ob/ob (stock #000632, JacksonLabs, Bar Harbor, Me.) and db/db (stock #000642, Jackson Labs, BarHarbor, Me.) mice respectively. Ob/ob and db/db mice are homozygousknockout mice for the leptin (ob) or leptin receptor (db) genes. Bothob/ob and db/db mice were developed in a C57BL/6J background strain andboth genetic alterations leads to spontaneous development of obesity by4-5 weeks of age when fed a standard laboratory chow diet.

5-week-old male ob/ob (n=6) and db/db (n=6) mice were housed using a 12h light dark cycle and received ad libitum access to a standardlaboratory chow-fed diet (Con) with 13% kcal fat, (diet #5053, Lab Diet,Inc, St. Louis, Mo.) and drinking water. After one week ob/ob and db/dbmice were randomly divided to receive either: i) con diet and drinkingwater containing 1% DMSO (n=3); or ii) con diet with drinking watercontaining 3.0 mg/100 ml AC261066. All mice remained on their diets for8 weeks. After 8 weeks all mice were subjected to metabolic studies andthen sacrificed by cervical dislocation and tissues were snap frozen at−70°C. for future RNA isolation and histology.

Metabolic Measurements-Glucose tolerance was performed using anintraperitoneal glucose tolerance test (GTT) as previously described(Trasino et al., Vitamin A Deficiency Causes Hyperglycemia and Loss ofPancreatic β-Cell Mass, J Biol Chem. 2014 Dec. 1. pii: jbc.M114.616763).Mice were fasted overnight followed by intra-peritoneal injections(n=3-5 per group) of 50% glucose in PBS at 2.0 g of glucose/kg of bodyweight. Tail vein blood was collected at 15, 30, 45, 60, and 120 minutespost-injection for glucose measurements using a FreeStyle Lite BloodGlucose Monitoring System (Abbott Diabetes Care, Inc. Alameda, Calif.).Insulin tolerance tests (ITT) were performed as previously described(Trasino et al., Vitamin A Deficiency Causes Hyperglycemia and Loss ofPancreatic β-Cell Mass, J Biol Chem. 2014 Dec. 1. pii: jbc.M114.616763).Mice were fasted for 4 hours followed by intra-peritoneal injectionswith insulin (Humulin R; Eli Lilly, 2 U/kg of body weight). Tail veinblood glucose was measured at 20, 40, 60 and 120 minutes after injectionusing a FreeStyle Lite Blood Glucose Monitoring System (Abbott DiabetesCare, Inc. Alameda, Calif.). To determine insulin secretion responses toglucose, serum fractions were isolated between 0-60 minutes post glucoseinjections and insulin concentrations were measured using anUltrasensitive Insulin ELISA Kit (Alpco, Inc. Salem, N.H.). Random bloodglucose measurements were taken from tail veins of 4 mice per group at2-3 random time points daily Means are expressed as±standard error ofthe mean (S.E.M) and P-values were calculated using one-way analysis ofvariance followed by Bonferroni post-hoc analysis.

Immunofluorescence and Immunostaining Microscopy-Paraffin embeddedpancreatic tissue sections were incubated with antibodies against:insulin (mouse monoclonal 1:300, #1061, Beta Cell Biology Consortium).We utilized Alexa-fluor 488 conjugated anti-mouse secondary antibody(1:500) (Invitrogen, Carlsbad, Calif.) for immunofluorescence labelingof insulin followed by visualization using a Nikon TE2000 invertedfluorescence microscope (Nikon, Inc).

Pancreatic Insulin Measurements-Pancreatic insulin levels were measuredin lysates from pancreatic tissues using an ultrasensitive Insulin ELISAKit (Alpco, Inc. Salem, N.H.) as per the manufacturers' instructions.Insulin concentrations were normalized to pancreatic proteinconcentrations determined using the DC protein assay (Bio-Rad, Inc.Hercules, Calif.) according to the manufacturers' protocol. Endocrinehormones levels are reported as mean±standard error of the mean (S.E.M)and P-values calculated using one-way analysis of variance followed byBonferroni multiple comparison test post-hoc analysis.

RNA Isolation and cDNA Synthesis-Total RNA was isolated from wholepancreas and small intestine homogenates using RNeasy mini kits (Qiagen,Valencia, Calif.) and quantified using a Nano Drop 2000spectrophotometer (Thermo Scientific, Wilmington, Del.). Total RNA (2μg) was used to synthesize cDNA with random primers using a qScript cDNAsynthesis kit (Quanta Biosciences, Gaithersburg, Md.).

Measurement of Pancreatic Endocrine Cell Mass.—Pancreatic endocrine cellmass was determined using a direct point counting method as previouslydescribed. Between 100-200 insulin positive fields per mouse werephotographed using a using a Nikon TE2000 inverted fluorescencemicroscope (Nikon, Inc) and analyzed for β-cell by using the followingformula: β-cell mass (mg)=total insulin positive islet area (μm²)/totalpancreatic tissue area (μm²)×pancreatic tissue weight (mg). Endocrinecell mass is reported as mean±standard error of the mean (S.E.M) andP-values were calculated using one-way analysis of variance followed byBonferroni multiple comparison test post-hoc analysis.

Tissue Triglyceride Analysis: Total tissue lipids were extracted fromusing the Folch method. Briefly, total lipids were extracted fromaliquots of tissue homogenates using chloroform: methanol (2:1) andpartitioned using dH2O. Organic phase solvents containing lipids wereevaporated under nitrogen gas and re-suspended in 0.5% (v/v) TritonX-100 solution in water. Tissue triglycerides were determinedenzymatically using Triglycerides Reagent kit (Invitrogen, LifeTechnologies, Carlsbad, Calif., USA) according to the manufacture'sprotocol. Tissue triglycerides were normalized to tissue proteinconcentrations and reported as mean±standard error of the mean (S.E.M)and P-values were calculated using one-way analysis of variance followedby Bonferroni multiple comparison test post-hoc analysis.

Quantitative RT-PCR (Q-PCR)-Q-PCR was performed using SYBR Green PCRmaster mix on a Bio-Rad MyiQ2 Real Time PCR iCycler (Bio-Rad, Inc.Hercules, Calif.). Gene specific primers (Table 5) were used to amplifymRNA target genes, which were normalized to Hprt internal control genes.cDNA from 3-5 mice per experimental group was analyzed for relative mRNAfold changes, calculated using the Pfaffl method (Pfaffl M W, A newmathematical model for relative quantification in real-time RT-PCR,Nucleic Acids Res. 2001 May 1; 29(9):e45). Relative gene expressionvalues are reported as mean±standard error of the mean (S.E.M) andP-values calculated using one-way analysis of variance followed byBonferroni multiple comparison test post-hoc analysis.

TABLE 5 Gene Expression Primers NCBI Gene Gene Forward PrimerReverse Primer Symbol Gene Name ID (5′---3′) (5′---3′)Pathway: Lipogenesis ACC Acetyl--- 107476 ATGGGCGGAATGGTCTCTGGGGACCTTGTCTTCATC 1 Coenzyme TTTC (SEQ ID NO. 61) AT (SEQ ID NO. 62) Acarboxylase alpha DGA Diacylglycc 13350 ATGATGGCTCAGGTCCCCACTGGGGCATCGTAGTT T1 rol O--- ACT (SEQ ID NO. 63) GA (SEQ ID NO. 64)acyltransfer ase 1 FAB Fatty Acid 11770 TGAAATCACCGCAGACGACACATTCCACCACCAGC P4 Binding ACA (SEQ ID NO. 65) TT (SEQ ID NO. 66)Protein 4 FAS Fatty Acid 14104 GGAGGTGGTGATAGCCG TGGGTAATCCATAGAGCC NSynthase GTAT (SEQ ID NO. 67) CAG(SEQ ID NO. 68) HMG 3--- 15357AGCTTGCCCGAATTGTA TCTGTTGTGAACCATGTG CR Hydroxy--- TGTG (SEQ ID NO. 69)ACTTC (SEQ ID NO. 70) 3--- Methyl glutaryl--- coenzyme A reductase PPAPeroxisome 19016 CTCCAAGAATACCAAAG GCCTGATGCTTTATCCCCA Rγproliferator--- TGCGA (SEQ ID NO. 71) CA (SEQ ID NO. 72) activatedreceptor gamma SCD Stearoyl--- 20249 GCTCTACACCTGCCTCTTCAGCCGAGCCTTGTAAGT 1 Coenzyme CG (SEQ ID NO. 73) TC (SEQ ID NO. 74) Adesaturase 1 SRE Sterol 20787 CAAGGCCATCGACTACA CACCACTTCGGGTTTCATG BP1regulatory TCCG (SEQ ID NO. 75) (SEQ ID NO. 76) element bindingtranscription factor 1 Pathway: Lipid β---Oxidation ACA Acetyl--- 110446AGCCTTTCGCGTCTCCAT TGCATAACTTCGTTCCAG T1 Coenzyme (SEQ ID NO. 77)GC (SEQ ID NO. 78) A acetyl transferase 1 CPT1 Carnitine 12894GCCCATGTTGTACAGCT AGTGGCCTCACAGACTCC α palmitoyl TCC (SEQ ID NO. 79)AG (SEQ ID NO. 80) transferase 1a CPT2 Carnitine 12896 CAGCACAGCATCGTACCTCCCAATGCCGTTCTCAA palmitoyl CA (SEQ ID NO. 81) AAT (SEQ ID NO. 82)transferase 2 MCD malonyl--- 56690 GCACGTCCGGGAAATGA GCCTCACACTCGCTGATCTCoA AC (SEQ ID NO. 83) T (SEQ ID NO. 84) decarboxylase PDK Pyruvate228026 GTTTATCCCCCGATTCAG TTACTCAGTGGAACACCG 1 dehydrogenaseGT (SEQ ID NO. 85) CC (SEQ ID NO. 86) kinase, isoenzyme 1 PDK Pyruvate27273 AGTGAACACTCCTTCGG TGACAGGGCTTTCTGGTCT 4 dehydrogenaseTGC (SEQ ID NO. 87) T (SEQ ID NO. 88) kinase, isoenzyme 4 PPA Peroxisome19013 AGAGCCCCATCTGTCCT ACTGGTAGTCTGCAAAAC Ra proliferatorCTC (SEQ ID NO. 89) CAAA(SEQ ID NO. 90) ---activatcd receptor alphaPathway: Adipocyte Metabolism ADIP Adiponectin 11450 TGTTCCTCTTAATCCTGCCCAACCTGCACAAGTTCC OQ CCA (SEQ ID NO. 91) CTT (SEQ ID NO. 92) PLINPerilipin1 103968 TGAAGCAGGGCCACTCT GACACCACCTGCATGGCT 1C (SEQ ID NO. 93) (SEQ ID NO. 94) HSL Hormone 16890 GATTTACGCACGATGACACCTGCAAAGACATTAGA sensitive ACAGT (SEQ ID NO. 95) CAGC (SEQ ID NO. 96)lipase UCP Uncoupling 22227 GTGAACCCGACAACTTC TGCCAGGCAAGCTGAAAC 1protein 1 CGAA (SEQ ID NO. 97) TC (SEQ ID NO. 98)Pathway: Inflammation and Fibrosis MCP Monocyte 20296 TTAAAAACCTGGATCGGGCATTAGCTTCAGATTTAC -1 chemo AACCAA (SEQ ID NO. 99)GGGT (SEQ ID NO. 100) attractant protein-1 TNF- Tumor 21926CCTGTAGCCCACGTCGT GGGAGTAGACAAGGTACA α necrosis AG (SEQ ID NO. 101)ACCC (SEQ ID NO. 102) factor-alpha α- alpha- 11475 GTCCCAGACATCAGGGATCGGATACTTCAGCGTCA SMA Smooth GTAA (SEQ ID NO. 103) GGA (SEQ ID NO. 104)muscle actin Pathway: Housekeeping Reference Gene HPR Hypoxanthine 15452GCTTGCTGGTGAAAAGG CCCTGAAGTACTCATTAT T guanine ACCTCTCGAAG (SEQ IDAGTCAAGGGCAT (SEQ ID phosphoribosyl NO. 105) NO. 106) transferase

Example 22—Retinoic Acid Receptor β (RARβ) Agonists Diminish DietInduced Body Weight Increases and Glucose Intolerance in a High FatModel of Diabetes

Three months of HF-fat (45% Kcal/fat) feeding led to a significantincrease in body weight compared to con-fed mice (FIG. 32A). Howevercompared to HF-fed mice, HF-fed mice treated with AC261066 or AC55649for 3 months had approximately a 10% decrease in body weights (FIG.32A). Metabolic studies demonstrated that water administration of theRARβ agonists AC261066 and AC55649 lead to improved glucose toleranceand area under the curve glucose in HF-fed mice (FIG. 32B, C). Comparedto con-fed mice, blood glucose levels HF-fed mice were unchanged afteran overnight fast suggesting impaired insulin signaling and peripheralinsulin resistance (FIG. 32D). HF-fed mice treated with AC261066 orAC55649 had significant reductions in overnight fasting glucose levels(FIG. 32D). Collectively these data demonstrate that administration ofthe specific RARβ agonists AC261066 and AC55649 for 3 months candecrease body weight and ameliorate impaired glucose tolerance in HF-fedobese mice. These studies suggest that AC261066 or AC55649 may also leadto improved hepatic and extra-hepatic glucose and insulin metabolismbased on the significant reductions in feed to fasting overnight glucoselevels.

METHODS. Preparation of RARβ agonists solution. AC261066 and AC55649were dissolved in dimethyl sulfoxide (DMSO) at the concentration of 3.0mg/ml and diluted in the drinking water for mice to the finalconcentration of 3.0-mg/100 ml. Mice, diet, and drug treatments. Wt maleC57/BL6 male mice were maintained on either a standard laboratorychow-fed diet (Con) with 13% kcal fat, (diet #5053, Lab Diet, Inc, St.Louis, Mo., [n=4]) or a high fat, western style diet (HFD) with 45%kcals from fat, (diet #58126, Lab Diet, Inc., St. Louis, Mo.) for 3months. Two weeks after the start of the HFD treatment, the HFD groupwas further split into 3 groups for 3 months: i) HFD and drinking watercontaining 1% DMSO (n=5); ii) high fat diet (HFD) and drinking watercontaining 3.0 mg/100 ml AC261066, a specific RARβ agonist (n=5) or iii)HFD and drinking water containing 3.0 mg/100 ml AC55649, a specific RARβagonist (n=4). After 3 months the mice were tested for glucoseintolerance with an intra-peritoneal glucose tolerance test (GTT). Micewere then sacrificed by cervical dislocation. Blood and various tissuesamples were harvested.

Glucose Tolerance Test (GTT). Glucose tolerance was performed using anintraperitoneal glucose tolerance test (GTT). Mice were fasted overnight(˜16 hrs) followed by intra-peritoneal injections (n=3-5 per group) of50% glucose in PBS at 2.0 g of glucose/kg of body weight. Tail veinblood was collected at 15, 30, 45, 60, and 120 minutes post-injectionfor glucose measurements using a FreeStyle Lite Blood Glucose MonitoringSystem (Abbott Diabetes Care, Inc. Alameda, Calif.). Random and fastingblood glucose measurements were taken from tail veins of 4 mice pergroup at 2-3 random time points daily or just prior to GTT. Means areexpressed as ±standard error of the mean (S.E.M) and P-values werecalculated using one-way analysis of variance followed by Bonferronipost-hoc analysis. Grubbs' maximum normal residual test was used fordetection of one or more outliers.

Example 23—Retinoic Acid Receptor β (RARβ) Agonist AC261066 Alters RenalExpression of Genes Involved in Lipid Metabolism and Inflammation inModels of Obesity and Diabetes

Diabetic kidney disease (aka diabetic nephropathy (DN)) frequentlyoccurs in individuals with type 2 diabetes. The causes of DN are unclearbut insulin resistance, hyperlipidemia and obesity are implicatedbecause these states can impair renal lipid metabolism leading to theaccumulation of free fatty acids (FFA), which can promote renalinflammation, fibrosis and impaired kidney function. There is evidencethat transcriptions factors involved in de novo lipid synthesis such assrebpl contribute to the pathogenesis of DN. Therefore we measuredkidney mRNA transcripts of genes involved in de novo lipid synthesis(SREBP1, FASN) and β-oxidation (catabolism) of lipids (PPAR-α andCPT1-α) and found that in comparison to wt con-fed mice, HFD-fed micehad a 4-5 fold increase in kidney mRNA levels of SREBP1 and FASN (FIG.33A). HF-fed mice treated with AC261066 had no increase in mRNAtranscripts of SREBP1 and FASN (FIG. 33A). We also found that incomparison to HFD-fed mice, mRNA transcripts of PPAR-α and CPT1-α wereincreased approximately 2-fold in HF-fed mice treated with AC261066(FIG. 33A).

We also measured kidney mRNA levels of pro-inflammatory and fibrogenicmediators MCP-1 (aka CCL2), TNF-α, and α-SMA in HFD-fed mice and twogenetic mouse models of obesity and diabetes, Ob/Ob and Db/Db mice.MCP-1, TNF-α, and α-SMA are implicated in inflammation and fibrosis inDN and expression of these genes are frequently elevated in models ofDN. Our PCR analysis results show that compared to wt con mice, HFD-fed,Ob/Ob, and Db/Db mice had a 4-8 fold and 20-30 fold increase in mRNAlevels of MCP-1 (FIG. 33B) and TNF-α (FIG. 33C,E) respectively (FIG.33B, 33C). Compared to HFD, Ob/Ob and Db/Db mice, we found that HFD,Ob/Ob and Db/Db mice treated with AC261066 had 40-50% reductions inkidney mRNA transcripts of MCP-1 (FIG. 33B) and TNF-α (FIG. 33C, E)respectively. Kidney mRNA transcripts of α-SMA, which contributes to DNfibrosis, was increased 5-6 fold in HFD Ob/Ob and Db/Db mice compared towt con (FIG. 33D). HFD Ob/Ob and Db/Db mice treated AC261066 had 40-50%reductions in a-SMA transcripts compared to their respective HFD andgenetic controls (FIG. 33D).

Retinoic Acid Receptor β (RARβ) Agonists Diminish The Activation ofFibrogenic Kidney Stellate Cells. Given that we observed a significantdecrease in mRNA transcripts of α-SMA we measured kidney proteinexpression of α-SMA in fibrogenic renal stellate cells Renal stellatecells (RSCs) are resident kidney fibroblasts, which in response toinflammation undergo differentiation to “activated” myofibroblasts thatsecrete contractile proteins such as α-SMA. Quiescent RSCs do notexpress α-SMA, but unchecked activation of RSCs and secretion of α-SMAcontributes to renal fibrosis and the pathogenesis of DN. RSCs expressthe mesenchymal protein marker vimentin. We used doubleimmunofluorescence to label RSCs and determine the percentage ofactivated RSCs expressing the fibrogenic protein α-SMA. Using Db/Dbmice, which spontaneously diabetes and DN in a similar manner to humans,we detected more than a 4-fold increase in activated α-SMA positive RSCscompared to wt con mice (FIG. 33F, G). Db/Db mice treated with AC261066for 4 weeks had more than a 50% reduction in α-SMA positive RSCs (FIG.33F, G).

Coupled with our PCR analysis of genes involved in lipid metabolism,inflammation and fibrogenesis, our immunofluorescence studiesdemonstrate in three dietary and genetic models of obesity and type 2diabetes that treatment with the RARβ agonist AC261066 led to kidneygene and protein expression patterns consistent with a decrease inkidney lipotoxicity, inflammation and fibrogenesis and risk fordeveloping DN.

METHODS. Immunofluorescence and Immunostaining Microscopy-Paraffinembedded pancreatic tissue sections were incubated with antibodiesagainst: insulin (mouse monoclonal 1:300, #1061, Beta Cell BiologyConsortium), vimentin (rabbit polyclonal 1:500, Santa Cruz), or α-SMA(mouse monoclonal, 1:1000, Dako, Inc).

We utilized Alexa-fluor 488 conjugated anti-mouse secondary antibody(1:500) (Invitrogen, Carlsbad, Calif.) for immunofluorescence labelingof insulin followed by visualization using a Nikon TE2000 invertedfluorescence microscope (Nikon, Inc).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices and materials are herein described. All publications mentionedherein are hereby incorporated by reference in their entirety for thepurpose of describing and disclosing the materials and methodologiesthat are reported in the publication which might be used in connectionwith the invention.

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1. A method of controlling cholesterol level and/or triglyceride levelin a subject in need thereof, comprising administering to said subjectvitamin A or a retinoic acid receptor-beta (RARβ) agonist.
 2. (canceled)3. A method of controlling glucose level, glucose intolerance, glucagonlevel and/or insulin sensitivity in a subject in need thereof,comprising administering to said subject vitamin A or a retinoic acidreceptor-beta (RARβ) agonist. 4.-7. (canceled)
 8. A method of treatingor preventing fat accumulation in a subject in need thereof comprisingadministering to said subject vitamin A or a retinoic acid receptor-beta(RARβ) agonist. 9.-10. (canceled)
 11. The method according to claim 1,wherein administration of said retinoic acid receptor-beta (RARβ)agonist treats or prevents a disease selected from the group consistingof a pancreatic disease, a cardiovascular disease, a liver disease, akidney disease, obesity, fibrosis, hyperlipidemia, hypertriglyceridemia,hyperglycemia, or an organ-specific vitamin A deficiency.
 12. The methodof claim 11, wherein said pancreatic disease is diabetes.
 13. The methodof claim 11, wherein said liver disease is fatty liver disease (FLD),non-alcoholic steatohepatitis (NASH), liver fibrosis, or hepaticsteatosis.
 14. The method of claim 11, wherein said kidney disease isdiabetic nephropathy.
 15. The method of claim 11, wherein said diseaseis associated with a high fat diet. 16.-17. (canceled)
 18. The methodaccording to claim 1, wherein said retinoic acid receptor-beta (RARβ)agonist is AC201066, AC55649 or a combination thereof.
 19. (canceled)20. The method according to claim 1, wherein said a retinoic acidreceptor-beta (RARβ) agonist is administered three times daily.
 21. Themethod according to claim 1, wherein said retinoic acid receptor-beta(RARβ) agonist is administered at an amount from about 30 mg to about200 mg per day.
 22. The method according to claim 1, wherein saidretinoic acid receptor-beta (RARβ) agonist is administered at aconcentration from about 0.1 mg to about 10 mg per 100 ml.
 23. Themethod according to claim 1, wherein said retinoic acid receptor-beta(RARβ) agonist is administered at a concentration from about 1 mg toabout 3 mg per 100 ml.
 24. The method according to claim 1, wherein saidretinoic acid receptor-beta (RARβ) agonist is administered orally. 25.The method according to claim 1, wherein said retinoic acidreceptor-beta (RARβ) agonist is administered intravenously orsubcutaneously.
 26. The method according to claim 1, further comprisingadministering a second drug. 27.-37. (canceled)